Methods for detection of a target nucleic acid by capture

ABSTRACT

The invention relates to a method of generating a signal indicative of the presence of a target nucleic acid in a sample, where the method includes forming a cleavage structure by incubating a sample comprising a target nucleic acid with a probe having a secondary structure that changes upon binding of the probe to the target nucleic acid and further comprising a binding moiety. The invention also includes the steps of cleaving the cleavage structure with a nuclease to release a nucleic acid fragment to generate a signal, wherein generation of the signal is indicative of the presence of a target nucleic acid in a sample, and detecting and/or measuring the amount of the fragment captured by binding of a binding moiety to a capture element on a solid support.  
     The invention also relates to a method of detecting or measuring a target nucleic acid in a sample, where the method includes forming a cleavage structure by incubating a sample containing a target nucleic acid with a probe having a secondary structure that changes upon binding of the probe to a target nucleic acid and comprising a binding moiety, and cleaving the cleavage structure with a nuclease to generate a cleaved nucleic acid fragment and detecting and/or measuring the amount of the fragment captured by binding of a binding moiety to a capture element on a solid support.

FIELD OF THE INVENTION

[0001] The invention relates in general to methods of detecting ormeasuring a target nucleic acid.

BACKGROUND OF THE INVENTION

[0002] The fidelity of DNA replication, recombination, and repair isessential for maintaining genome stability, and these processes dependon 5′→3′ exonuclease enzymes which are present in all organisms. For DNArepair, these enzymes are required for damaged fragment excision andrecombinational mismatch correction. For replication, these nucleasesare critical for the efficient processing of Okazaki fragments duringlagging strand DNA synthesis. In Escherichia coli, this latter activityis provided by DNA polymerase I (PolI); E. coli strains withinactivating mutations in the PolI 5′→3′ exonuclease domain are notviable due to an inability to process Okazaki fragments. Eukaryotic DNApolymerases, however, lack an intrinsic 5′→3′ exonuclease domain, andthis critical activity is provided by the multifunctional,structure-specific metallonuclease FEN-1 (five′ exonuclease-1 or flapendonuclease-1), which also acts as an endonuclease for 5′ DNA flaps(Reviewed in Hosfield et al., 1998a, Cell, 95:135).

[0003] Methods of detecting and/or measuring a nucleic acid wherein anenzyme produces a labeled nucleic acid fragment are known in the art.

[0004] U.S. Pat. Nos. 5,843,669, 5,719,028, 5,837,450, 5,846,717 and5,888,780 disclose a method of cleaving a target DNA molecule byincubating a 5′ labeled target DNA with a DNA polymerase isolated fromThermus aquaticus (Taq polymerase) and a partially complementaryoligonucleotide capable of hybridizing to sequences at the desired pointof cleavage. The partially complementary oligonucleotide directs the Taqpolymerase to the target DNA through formation of a substrate structurecontaining a duplex with a 3′ extension opposite the desired site ofcleavage wherein the non-complementary region of the oligonucleotideprovides a 3′ arm and the unannealed 5′ region of the substrate moleculeprovides a 5′ arm. The partially complementary oligonucleotide includesa 3′ nucleotide extension capable of forming a short hairpin either whenunhybridized or when hybridized to a target sequence at the desiredpoint of cleavage. The release of labeled fragment is detected followingcleavage by Taq polymerase.

[0005] U.S. Pat. Nos. 5,843,669, 5,719,028, 5,837,450, 5,846,717 and5,888,780 disclose the generation of mutant, thermostable DNApolymerases that have very little or no detectable synthetic activity,and wild type thermostable nuclease activity. The mutant polymerases aresaid to be useful because they lack 5′ to 3′ synthetic activity; thussynthetic activity is an undesirable side reaction in combination with aDNA cleavage step in a detection assay.

[0006] U.S. Pat. Nos. 5,843,669, 5,719,028, 5,837,450, 5,846,717 and5,888,780 disclose that wild type Taq polymerase or mutant Taqpolymerases that lack synthetic activity can release a labeled fragmentby cleaving a 5′ end labeled hairpin structure formed by heatdenaturation followed by cooling, in the presence of a primer that bindsto the 3′ arm of the hairpin structure. Further, U.S. Pat. Nos.5,843,669, 5,719,028, 5,837,450, 5,846,717 and 5,888,780 teach that themutant Taq polymerases lacking synthetic activity can also cleave thishairpin structure in the absence of a primer that binds to the 3′ arm ofthe hairpin structure.

[0007] U.S. Pat. Nos. 5,843,669, 5,719,028, 5,837,450, 5,846,717 and5,888,780 also disclose that cleavage of this hairpin structure in thepresence of a primer that binds to the 3′ arm of the hairpin structureby mutant Taq polymerases lacking synthetic activity yields a singlespecies of labeled cleaved product, while wild type Taq polymeraseproduces multiple cleavage products and converts the hairpin structureto a double stranded form in the presence of dNTPs, due to the highlevel of synthetic activity of the wild type Taq enzyme.

[0008] U.S. Pat. Nos. 5,843,669, 5,719,028, 5,837,450, 5,846,717 and5,888,780 also disclose that mutant Taq polymerases exhibiting reducedsynthetic activity, but not wild type Taq polymerase, can release asingle labeled fragment by cleaving a linear nucleic acid substratecomprising a 5′ end labeled target nucleic acid and a complementaryoligonucleotide wherein the complementary oligonucleotide hybridizes toa portion of the target nucleic acid such that 5′ and 3′ regions of thetarget nucleic acid are not annealed to the oligonucleotide and remainsingle stranded.

[0009] There is a need in the art for a method of generating a signalthat can be easily distinguished from oligonucleotide fragments that mayarise from nuclease contaminants, using a nucleic acid cleavagereaction.

[0010] There is a need in the art for a method of generating a signalthat utilizes a probe comprising secondary structure wherein some or allof the self-complementary regions of the probe that anneal to form thesecondary structure are melted when the probe hybridizes with a targetnucleic acid, thereby reducing non-specific binding of the probe to thetarget, and increasing the specificity of the assay.

[0011] U.S. Pat. Nos. 5,843,669, 5,719,028, 5,837,450, 5,846,717 and5,888,780 also disclose a method of cleaving a labeled nucleic acidsubstrate at naturally occurring areas of secondary structure. Accordingto this method, biotin labeled DNA substrates are prepared by PCR, mixedwith wild type Taq polymerase or CleavaseBN (a mutant Taq polymerasewith reduced synthetic activity and wild type 5′ to 3′ nucleaseactivity), incubated at 95° C. for 5 seconds to denature the substrateand then quickly cooled to 65° C. to allow the DNA to assume its uniquesecondary structure by allowing the formation of intra-strand hydrogenbonds between the complementary bases. The reaction mixture is incubatedat 65° C. to allow cleavage to occur and biotinylated cleavage productsare detected.

[0012] There is a need in the art for a method of generating a signalusing a nucleic acid cleavage reaction wherein the cleavage structure isnot required to contain areas of secondary structure.

[0013] Methods of detecting and/or measuring a nucleic acid wherein aFEN-1 enzyme is used to generate a labeled nucleic acid fragment areknown in the art.

[0014] U.S. Pat. No. 5,843,669 discloses a method of detectingpolymorphisms by cleavase fragment length polymorphism analysis using athermostable FEN-1 nuclease in the presence or absence of a mutant Taqpolymerase exhibiting reduced synthetic activity. According to thismethod, double stranded Hepatitis C virus (HCV) DNA fragments arelabeled by using 5′ end labeled primers (labeled with TMR fluorescentdye) in a PCR reaction. The TMR labeled PCR products are denatured byheating to 95° C. and cooled to 55° C. to generate a cleavage structure.U.S. Pat. No. 5,843,669 discloses that a cleavage structure comprises aregion of a single stranded nucleic acid substrate containing secondarystructure. Cleavage is carried out in the presence of CleavaseBNnuclease, FEN-1 nuclease derived from the archaebacteria Methanococcusjannashii or both enzymes. Labeled reaction products are visualized bygel electrophoresis followed by fluoroimaging. U.S. Pat. No. 5,843,669discloses that CleavaseBN nuclease and Methanococcus jannaschii FEN-1nuclease produce cleavage patterns that are easily distinguished fromeach other, and that the cleavage patterns from a reaction containingboth enzymes include elements of the patterns produced by cleavage witheach individual enzyme but are not merely a composite of the cleavagepatterns produced by each individual enzyme. This indicates that some ofthe fragments that are not cleaved by one enzyme (and which appear as aband in that enzyme's pattern) can be cleaved by a second enzyme in thesame reaction mixture.

[0015] Lyamichev et al. disclose a method for detecting DNAs whereinoverlapping pairs of oligonucleotide probes that are partiallycomplementary to a region of target DNA are mixed with the target DNA toform a 5′ flap region, and wherein cleavage of the labeled downstreamprobe by a thermostable FEN-1 nuclease produces a labeled cleavageproduct. Lyamichev et al. also disclose reaction conditions whereinmultiple copies of the downstream oligonucleotide probe can be cleavedfor a single target sequence in the absence of temperature cycling, soas to amplify the cleavage signal and allow quantitative detection oftarget DNA at sub-attomole levels (Lyamichev et al., 1999, Nat.Biotechnol., 17:292).

[0016] The polymerase chain reaction (PCR) technique, is disclosed inU.S. Pat. Nos. 4,683,202, 4,683,195 and 4,800,159. In its simplest form,PCR is an in vitro method for the enzymatic synthesis of specific DNAsequences, using two oligonucleotide primers that hybridize to oppositestrands and flank the region of interest in the target DNA. A repetitiveseries of reaction steps involving template denaturation, primerannealing and the extension of the annealed primers by DNA polymeraseresults in the exponential accumulation of a specific fragment whosetermini are defined by the 5′ ends of the primers. PCR is reported to becapable of producing a selective enrichment of a specific DNA sequenceby a factor of 10 ⁹. The PCR method is also described in Saiki et al.,1985, Science, 230:1350.

[0017] While the PCR technique is an extremely powerful method foramplifying nucleic acid sequences, the detection of the amplifiedmaterial requires additional manipulation and subsequent handling of thePCR products to determine whether the target DNA is present. It isdesirable to decrease the number of subsequent handling steps currentlyrequired for the detection of amplified material. An assay system,wherein a signal is generated while the target sequence is amplified,requires fewer handling steps for the detection of amplified material,as compared to a PCR method that does not generate a signal during theamplification step.

[0018] U.S. Pat. Nos. 5,210,015 and 5,487,972 disclose a PCR based assayfor releasing labeled probe comprising generating a signal during theamplification step of a PCR reaction in the presence of a nucleic acidto be amplified, Taq polymerase that has 5′ to 3′ exonuclease activityand a 5′, 3′ or 5′ and 3′ end-labeled probe comprising a regioncomplementary to the amplified region and an additionalnon-complementary 5′ tail region. U.S. Pat. Nos. 5,210,015 and 5,487,972disclose further that this PCR based assay can liberate the 5′ labeledend of a hybridized probe when the Taq polymerase is positioned near thelabeled probe by an upstream probe in a polymerization independentmanner, e.g. in the absence of dNTPs.

[0019] There is a need in the art for a method of detecting or measuringa target nucleic acid that does not require multiple steps.

[0020] There is also a need in the art for a PCR process for detectingor measuring a target nucleic acid that does not require multiple stepssubsequent to the amplification process.

[0021] There is also a need in the art for a PCR process for detectingor measuring a target nucleic acid that allows for concurrentamplification and detection of a target nucleic acid in a sample.

SUMMARY OF THE INVENTION

[0022] The invention provides a method of generating a signal indicativeof the presence of a target nucleic acid in a sample, which includes thesteps of forming a cleavage structure by incubating a sample containinga target nucleic acid with a probe containing a binding moiety andhaving a secondary structure that changes upon binding of the probe to atarget nucleic acid, and cleaving the cleavage structure with a nucleaseto release a nucleic acid fragment and thus generate a signal. Nucleasecleavage of the cleavage structure occurs at a cleaving temperature, andthe secondary structure of the probe when not bound to the targetnucleic acid is stable at or below the cleaving temperature. Generationof the signal is indicative of the presence of a target nucleic acid inthe sample, and the signal is detected or measured by detecting and/ormeasuring the amount of the fragment captured by binding of the bindingmoiety to a capture element on a solid support.

[0023] As used herein, a “probe” refers to a single stranded nucleicacid comprising a region or regions that are complementary to a targetnucleic acid (e.g., target nucleic acid binding sequences) (for exampleC in FIG. 4). A “probe” according to the invention has a secondarystructure that changes upon binding of the probe to the target nucleicacid and further comprises a binding moiety. A “probe” according to theinvention binds to a target nucleic acid to form a cleavage structurethat can be cleaved by a nuclease, wherein cleaving is performed at acleaving temperature, and wherein the secondary structure of the probewhen not bound to the target nucleic acid is, preferably, stable at orbelow the cleaving temperature. A probe according to the inventioncannot be cleaved to generate a signal by a “nuclease”, as definedherein, prior to binding to a target nucleic acid. In one embodiment ofthe invention, a probe may comprise a region that cannot bind or is notcomplementary to a target nucleic acid. In another embodiment of theinvention, a probe does not have a secondary structure when bound to atarget nucleic acid.

[0024] As used herein, “secondary structure” refers to athree-dimensional conformation (for example a hairpin, a stem-loopstructure, an internal loop, a bulge loop, a branched structure or apseudoknot, FIGS. 1 and 3; multiple stem loop structures, cloverleaftype structures or any three dimensional structure. As used herein,“secondary structure” includes tertiary, quaternary etc . . . structure.A probe comprising such a three-dimensional structure binds to a targetnucleic acid to form a cleavage structure that can be cleaved by anuclease at a cleaving temperature. The three dimensional structure ofthe probe when not bound to the target nucleic acid is, preferably,stable at or below the cleaving temperature. “Secondary structure” asused herein, can mean a sequence comprising a first single-strandedsequence of bases (referred to herein as a “complementary nucleic acidsequence” (for example b in FIG. 4)) followed by a second complementarysequence either in the same molecule (for example b′ in FIG. 4), or in asecond molecule comprising the probe, folds back on itself to generatean antiparallel duplex structure, wherein the single-stranded sequenceand the complementary sequence (that is, the complementary nucleic acidsequences) anneal by the formation of hydrogen bonds. Oligonucleotideprobes, as used in the present invention include oligonucleotidescomprising secondary structure, including, but not limited to molecularbeacons, safety pins (FIG. 9), scorpions (FIG. 10), andsunrise/amplifluor probes (FIG. 11), the details and structures of whichare described below and in the corresponding figures.

[0025] As used herein, first and second “complementary” nucleic acidsequences are complementary to each other and can anneal by theformation of hydrogen bonds between the complementary bases.

[0026] A secondary structure also refers to the conformation of anucleic acid molecule comprising an affinity pair, defined herein,wherein the affinity pair reversibly associates as a result ofattractive forces that exist between the pair of moieties comprising theaffinity pair. As used herein, secondary structure prevents the bindingmoiety on the probe from binding to a capture element, and a change insecondary structure upon binding of the probe to the target nucleic acidand subsequent cleavage of the bound probe permits the binding moiety tobe captured by the capture element.

[0027] A “probe” according to the invention can be unimolecular. As usedherein, a “unimolecular” probe comprises a single molecule that binds toa target nucleic acid to form a cleavage structure that can be cleavedby a nuclease, wherein cleaving is performed at a cleaving temperature,and wherein the secondary structure of the “unimolecular” probe when notbound to the target nucleic acid is, preferably, stable at or below thecleaving temperature. Unimolecular probes useful according to theinvention include but are not limited to beacon probes, probescomprising a hairpin, stem-loop, internal loop, bulge loop or pseudoknotstructure, scorpion probes and sunrise/amplifluor probes.

[0028] A “probe” according to the invention can be more than onemolecule (e.g., bi-molecular or multi-molecular). At least one of themolecules comprising a bi-molecular or multi-molecular probe binds to atarget nucleic acid to form a cleavage structure that can be cleaved bya nuclease, wherein cleaving is performed at a cleaving temperature, andwherein the secondary structure of the molecule of the probe when notbound to the target nucleic acid is, preferably, stable at or below thecleaving temperature. The molecules comprising the multimolecular probeassociate with each other via intermolecular bonds (e.g., hydrogen bondsor covalent bonds). For example, a heterologous loop (see FIG. 1), or acloverleaf structure wherein one or more loops of the cloverleafstructure comprises a distinct molecule, and wherein the molecules thatassociate to form the cloverleaf structure associate via intermolecularbonding (e.g., hydrogen bonding or covalent bonding), are examples ofmultimolecular probes useful according to the invention.

[0029] As used herein, a “molecule” refers to a polynucleotide, andincludes a polynucleotide further comprising an attached member ormembers of an affinity pair.

[0030] A “probe” or a “molecule” comprising a probe is 5-10,000nucleotides in length, ideally from 6-5000, 7-1000, 8-500, 9-250, 10-100and 17-40 nucleotides in length, although probes or a moleculecomprising a probe of different lengths are useful.

[0031] A “probe” according to the invention has a target nucleic acidbinding sequence that is from 5 to 10,000 nucleotides, and preferablyfrom 10 to about 140 nucleotides. A “probe” according to the inventioncomprises at least first and second complementary nucleic acid sequencesor regions that are 3-250, preferably 4-150, and more preferably 5-110and most preferably 6-50 nucleotides long. The first and secondcomplementary nucleic acid sequences may have the same length or may beof different lengths. The invention provides for a probe wherein thefirst and second complementary nucleic acid sequences are both locatedupstream (5′) of the target nucleic acid binding site. Alternatively,the first and second complementary nucleic acid sequences can both belocated downstream (3′) of the target nucleic acid binding site. Inanother embodiment, the invention provides for a probe wherein the firstcomplementary nucleic acid sequence is upstream (5′) of the targetnucleic acid binding site and the second complementary nucleic acidsequence is downstream (3′) of the target nucleic acid binding site. Inanother embodiment, the invention provides for a probe wherein thesecond complementary nucleic acid sequence is upstream (5′) of thetarget nucleic acid binding site and the first complementary nucleicacid sequence is downstream (3′) of the target nucleic acid bindingsite. The actual length will be chosen with reference to the targetnucleic acid binding sequence such that the secondary structure of theprobe is, preferably, stable when the probe is not bound to the targetnucleic acid at the temperature at which cleavage of a cleavagestructure comprising the probe bound to a target nucleic acid isperformed. As the target nucleic acid binding sequence increases in sizeup to 500 nucleotides, the length of the complementary nucleic acidsequences may increase up to 15-125 nucleotides. For a target nucleicacid binding sequence greater than 100 nucleotides, the length of thecomplementary nucleic acid sequences need not be increased further. Ifthe probe is also an allele-discriminating probe, the lengths of thecomplementary nucleic acid sequences are more restricted, as isdiscussed below.

[0032] As used herein, the “target nucleic acid binding sequence” refersto the region of the probe that binds specifically to the target nucleicacid.

[0033] A “hairpin structure” or a “stem” refers to a double-helicalregion formed by base pairing between adjacent, inverted, complementarysequences in a single strand of RNA or DNA.

[0034] A “stem-loop” structure refers to a hairpin structure, furthercomprising a loop of unpaired bases at one end.

[0035] As used herein, a probe with “stable” secondary structure whennot bound to a target nucleic acid, refers to a secondary structurewherein 50% or more (e.g., 50%, 55%, 75% or 100%) of the base pairs thatconstitute the probe are not dissociated under conditions which permithybridization of the probe to the target nucleic acid, but in theabsence of the target nucleic acid.

[0036] “Stability” of a nucleic acid duplex is determined by the meltingtemperature, or “T_(m)”. The T_(m) of a particular nucleic acid duplexunder specified conditions (e.g., salt concentration and/or the presenceor absence of organic solvents) is the temperature at which half (50%)of the base pairs of the duplex molecule have disassociated (that is,are not hybridized to each other in a base-pair).

[0037] The “stability” of the secondary structure of a probe when notbound to the target nucleic acid is defined in a melting temperatureassay, in a fluorescence resonance energy transfer (FRET) assay or in afluorescence quenching assay, (the details or which are described in asection entitled, “Determining the Stability or the Secondary Structureof a Probe”).

[0038] A probe useful in the invention preferably will have secondarystructure that is “stable”, when not bound to a target, at or below thetemperature of the cleavage reaction. Thus, the temperature at whichnuclease cleavage of a probe/target nucleic acid hybrid is performedaccording to the invention, must be lower than the Tm of the secondarystructure. The secondary structure of the probe is “stable” in a meltingtemperature assay at a temperature that is at or below the temperatureof the cleavage reaction (i.e., at which cleavage is performed) if thelevel of light absorbance at the temperature at or below the temperatureof the cleavage reaction is less than (i.e., at least 5% less than,preferably 20% less than and most preferably 25% less than etc . . . )than the level of light absorbance at a temperature that is equal to orgreater than the Tm of the probe.

[0039] According to the method of the invention, the stability of asecondary structure can be measured by a FRET assay or a fluorescencequenching assay (described in the section entitled, “Determining theStability of the Secondary Structure of a Probe”). As used herein, afluorescence quenching assay can include a FRET assay. A probe accordingto the invention is labeled with an appropriate pair of interactivelabels (e.g., a FRET pair (for example as described in the sectionentitled, “Determining the Stability of the Secondary Structure of theProbe”, below) that can interact over a distance of, for example 2nucleotides, or a non-FRET-pair, (e.g., tetramethylrhodamine and DABCYL)that can interact over a distance of, for example, 20 nucleotides. Forexample, a probe according to the invention may be labeled with afluorophore and a quencher and fluorescence is then measured, in theabsence of a target nucleic acid, at different temperatures. The Tm isthe temperature at which the level of fluorescence is 50% of the maximallevel of fluorescence observed for a particular probe, see FIG. 12e. TheTm for a particular probe wherein the nucleic acid sequence of the probeis known, can be predicted according to methods known in the art. Thus,fluorescence is measured over a range of temperatures, e.g., wherein thelower temperature limit of the range is at least 50° Celsius below, andthe upper temperature limit of the range is at least 50° Celsius abovethe Tm or predicted Tm, for a probe according to the invention.

[0040] A secondary structure is herein defined as “stable” in a FRETassay at a temperature that is at or below the cleaving temperature ifthe level or wavelength of fluorescence is increased or decreased (e.g.,at least 5% less than, preferably 20% less than and more preferably 25%less than, etc . . . ) as compared with the level or wavelength of FRETthat is observed at the Tm of the probe (see FIGS. 12e and f). Forexample, an increase or a decrease in FRET can occur in a FRET assayaccording to the invention. In another embodiment, a shift inwavelength, which results in an increase in the new, shifted wavelengthor, a decrease in the new shifted wavelength, can occur in a FRET assayaccording to the invention.

[0041] A “change” in a secondary structure, according to the inventioncan be measured in a fluorescence quenching assay wherein a probeaccording to the invention comprises a fluorophore and a quencher thatare positioned such that in the absence of a target nucleic acid, and attemperatures below the Tm of the probe there is quenching of thefluorescence (as described above). As used herein, a “change” insecondary structure that occurs when a probe according to the inventionbinds to a target nucleic acid, refers to an increase in fluorescence insuch an assay, such that the level of fluorescence after binding of theprobe to the target nucleic acid at a temperature below the Tm of theprobe, is greater than (e.g., at least 5%, preferably 5-20% and mostpreferably 25% or more) the level of fluorescence observed in theabsence of a target nucleic acid (see FIG. 12g).

[0042] A secondary structure, according to the invention, can bedetected by subjecting a probe comprising a fluorophore and a quencherto a fluorescence quenching assay (as described above). A probe thatexhibits a change in fluorescence that correlates with a change intemperature, see FIG. 12e (e.g., fluorescence increases as thetemperature of the FRET reaction is increased) may be capable of forminga secondary structure.

[0043] As used herein, a “cleaving temperature” that is useful accordingto the invention is a temperature that is less than (at least 1° C. andpreferably 10° C.) the T_(m) of a probe having a secondary structure.The “cleaving temperature” is initially selected to be possible andpreferably optimal for the particular nuclease being employed in thecleavage reaction.

[0044] Preferably the 3′ terminus of the probe will be “blocked” toprohibit incorporation of the probe into a primer extension product ifan active polymerase is used in the reaction. “Blocking” can be achievedby using non-complementary bases or by adding a chemical moiety such asbiotin or a phosphate group to the 3′ hydroxl of the last nucleotide,which may, depending upon the selected moiety, serve a dual purpose byalso acting as a label for subsequent detection or capture of thenucleic acid attached to the label. Blocking can also be achieved byremoving the 3′-OH or by using a nucleotide that lacks a 3′-OH such asdideoxynucleotide.

[0045] The term probe encompasses an allele-discriminating probe. Asused herein, an “allele-discriminating” probe preferentially hybridizesto perfectly complementary target nucleic acids and discriminatesagainst sequences that vary by at least one nucleotide. A nucleic acidsequence which differs by at least one nucleotide, as compared to atarget nucleic acid, hereafter referred to as a “target-like nucleicacid sequence”, is thus not a target nucleic acid for anallele-discriminating probe according to the invention.Allele-discriminating probes do not hybridize sufficiently to atarget-like nucleic acid sequence that contains one or more nucleotidemismatches as compared to the target nucleic acid complementarysequence, at a particular temperature or within a range of temperaturesdetermined by experimental optimization to permit an allelediscriminating probe to discriminate between a target and a target-likesequence with at least a single nucleotide difference, and thus do notundergo a change in secondary structure upon binding to a target-likenucleic acid sequence in the presence of only a target-like nucleic acidsequence, and under conditions that would support hybridization of theallele discriminating probe to a target nucleic acid.

[0046] In one embodiment, an “allele-discriminating probe” according tothe invention refers to a probe that hybridizes to a target-like nucleicacid sequence that varies by at least one nucleotide from the targetnucleic acid, wherein the variant nucleotide(s) is/are not located inthe allele-discriminating site. According to this embodiment of theinvention, “an allele-discriminating probe” cannot bind to a target-likenucleic acid sequence that also varies by at least one nucleotide in theallele-discriminating site, at a particular temperature or within arange of temperatures determined by experimental optimization to permitan allele discriminating probe to discriminate between a target and atarget-like sequence with at least a single nucleotide difference.Single nucleotide differences only affect the percentage of a probe thatis bound to a target or target-like nucleic acid sequence. For example,the invention provides for a perfectly matched probe, wherein as much as100% of the target or is in a probe-target complex (e.g., is bound byprobe), in the presence of excess probe. The invention also provides forprobes comprising at least a single base mismatch wherein at least 1-5%and preferably 5-10% of the target-like sequence is bound by the probeunder the same conditions used to form a complex comprising a targetsequence and a perfectly matched probe.

[0047] As used herein, “allele-discriminating site” refers to a regionof a target nucleic acid that is different (i.e., by at least onenucleotide) from the corresponding region in all possible allelescomprising the target nucleic acid.

[0048] Allele-discriminating probes useful according to the inventionalso include probes that bind less effectively to a target-likesequence, as compared to a target sequence. The effectiveness of bindingof a probe to a target sequence or a target-like sequence can bemeasured in a FRET assay, performed at a temperature that is below (atleast 1° C. and preferably 10° C. or more) the Tm of the secondarystructure of the probe, in the presence of a target-like sequence or atarget sequence. The change in the level of fluorescence in the presenceor absence of a target sequence compared to the change in the level offluorescence in the presence or absence of a target-like sequence,provides an effective measure of the effectiveness of binding of a probeto a target or target-like sequence.

[0049] In a method according to the invention, a probe that binds lesseffectively to a target-like sequence as compared to a target sequencewould undergo a smaller (e.g., preferably 25-50%, more preferably 50-75%and most preferably 75-90% of the value of the change in fluorescenceupon binding to a target nucleic acid) change in secondary structure, asdetermined by measuring fluorescence in a FRET or fluorescence quenchingassay as described herein, upon hybridization to a target-like sequenceas compared to a target nucleic acid. In a method according to theinvention, a probe that binds less effectively to a target-like sequenceas compared to a target sequence would generate a signal that isindicative of the presence of a target-like nucleic acid sequence in asample. However, the intensity of the signal would be altered (e.g.,preferably 25-50%, more preferably 50-75% and most preferably 75-90%less than or more than the value of the change in fluorescence uponbinding to a target nucleic acid) the intensity of a signal generated inthe presence of a target sequence, as described hereinabove for asmaller change.

[0050] A “signal that is indicative of the presence of a target nucleicacid” or a “target-like nucleic acid sequence” refers to a signal thatis equal to a signal generated from 1 molecule to 10²⁰ molecules, morepreferably about 100 molecules to 10¹⁷ molecules and most preferablyabout 1000 molecules to 10¹⁴ molecules of a target nucleic acid or atarget-like nucleic acid sequence.

[0051] As used herein, a “binding moiety” refers to a region of a probe(for example ab in FIG. 4) that is released upon cleavage of the probeby a nuclease and binds specifically to a capture element as a result ofattractive forces that exist between the binding moiety and the captureelement, and wherein specific binding between the binding moiety and thecapture element only occurs when the secondary structure of the probehas “changed”, as defined herein. “Binds specifically” means viahydrogen bonding with a complementary nucleic acid or via an interactionbetween for example, the binding moiety and a binding protein capable ofbinding specifically to the nucleic acid sequence of the binding moiety.A “binding moiety” does not interfere with the ability of a probe tobind to a target nucleic acid. A binding moiety is incapable of bindingto a capture element when the probe is in its native secondarystructural conformation and that, upon binding to a target or templatenucleic acid, the secondary structure changes in a way that allows thebinding moiety to bind to the capture element, preferably after cleavageby a cleavage agent.

[0052] In one embodiment, the region of a probe that is cleaved to forma binding moiety cannot hybridize to a target nucleic acid. The regionof a “binding moiety” that is not a “complementary nucleic acidsequence”, as defined herein, (e.g., a in FIG. 4), is from 1-60nucleotides, preferably from 1-25 nucleotides and most preferably from1-10 nucleotides in length. Methods of detecting specific bindingbetween a binding moiety or a binding moiety, as defined herein, and acapture element, as defined herein, are well known in the art and aredescribed hereinbelow.

[0053] In one embodiment of the invention, a probe further comprises a“reporter”.

[0054] As used herein, a “reporter” refers to a “label”, definedhereinbelow and/or a “tag” defined hereinbelow.

[0055] As used herein, “label” or “labeled moiety capable of providing asignal” refers to any atom or molecule which can be used to provide adetectable (preferably quantifiable) signal, and which can beoperatively linked to a nucleic acid. Labels may provide signalsdetectable by fluorescence, radioactivity, colorimetry, gravimetry,X-ray diffraction or absorption, magnetism, enzymatic activity, massspectrometry, binding affinity, hybridization radiofrequency,nanocrystals and the like. A labeled probe according to the methods ofthe invention is labeled at the 5′ end, the 3′ end or internally. Thelabel can be “direct”, i.e. a dye, or “indirect”. i.e. biotin, digoxin,alkaline phosphatase,(AP), horse radish peroxidase (HRP) etc . . . Fordetection of “indirect labels” it is necessary to add additionalcomponents such as labeled antibodies, or enzyme substrates to visualizethe, captured, released, labeled nucleic acid fragment. In oneembodiment of the invention, a label cannot provide a detectable signalunless the secondary structure has “changed”, as defined herein, (forexample, such that the binding moiety is accessible).

[0056] A “binding moiety” also refers to a “tag”. As used herein, a“tag” refers to a moiety that is operatively linked to the 5′ end of aprobe (for example R in FIG. 1) and specifically binds to a captureelement as a result of attractive forces that exist between the tag andthe capture element, and wherein specific binding between the tag andthe capture element only occurs when the secondary structure of theprobe has changed (for example, such that the tag is accessible to acapture element). “Specifically binds” as it refers to a “tag” and acapture element means via covalent or hydrogen bonding or electrostaticattraction or via an interaction between, for example a protein and aligand, an antibody and an antigen, protein subunits, or a nucleic acidbinding protein and a nucleic acid binding site. A tag does notinterfere with the ability of a probe to anneal to a target nucleicacid. Tags include but are not limited to biotin, streptavidin, avidin,an antibody, an antigen, a hapten, a protein, or a chemically reactivemoiety. A “tag” as defined herein can bind to a “capture element” asdefined herein. According to the invention, a “tag” and a “captureelement” function as a binding pair. For example, in one embodiment, ifa tag is biotin, the corresponding capture element is avidin.Alternatively, in another embodiment, if a tag is an antibody, thecorresponding capture element is an antigen.

[0057] The invention contemplates a “probe” comprising a binding moiety,a “probe” comprising a “tag”, as defined herein, and a “probe”comprising both a binding moiety that is a region of a probe that isreleased upon cleavage of the probe by a nuclease (for example a nucleicacid sequence that binds to a capture element), and a “tag”.

[0058] As used herein, a “capture element” refers to a substance that isattached to a solid substrate for example by chemical crosslinking orcovalent binding, wherein the substance specifically binds to (e.g., viahydrogen bonding or via an interaction between, a nucleic acid bindingprotein and a nucleic acid binding site or between complementary nucleicacids) a binding moiety as a result of attractive forces that existbetween the binding moiety and the capture element, and wherein specificbinding between the binding moiety and the capture element only occurswhen the secondary structure of the probe comprising the binding moietyhas “changed”, as defined herein. Capture elements include but are notlimited to a nucleic acid binding protein or a nucleotide sequence.

[0059] As used herein, a “capture element” also refers to a substancethat is attached to a solid substrate for example by chemicalcrosslinking or covalent binding, wherein the substance specificallybinds to (e.g. via covalent or hydrogen bonding or electrostaticattraction via an interaction between, for example a protein and aligand, an antibody and an antigen, protein subunits, a nucleic acidbinding protein and a nucleic acid binding site or between complementarynucleic acids) a tag as a result of attractive forces that exist betweenthe tag and the capture element, and wherein specific binding betweenthe tag and the capture element only occurs when the secondary structureof the probe comprising the tag has “changed”, as defined herein.Capture elements include but are not limited to biotin, avidin,streptavidin, an antibody, an antigen, a hapten, a protein, or achemically reactive moiety. A “tag” as defined herein can bind to a“capture element” as defined herein. According to the invention, a “tag”and a “capture element” function as a binding pair. For example, in oneembodiment, if a capture element is biotin, the corresponding tag isavidin. Alternatively, in another embodiment, if a capture element is anantibody, the corresponding tag is an antigen.

[0060] As used herein, “solid support” means a surface to which amolecule (e.g. a capture element) can be irreversibly bound, includingbut not limited to membranes, sepharose beads, magnetic beads, tissueculture plates, silica based matrices, membrane based matrices, beadscomprising surfaces including but not limited to styrene, latex orsilica based materials and other polymers for example cellulose acetate,teflon, polyvinylidene difluoride, nylon, nitrocellulose, polyester,carbonate, polysulphone, metals, zeolites, paper, alumina, glass,polypropyle, polyvinyl chloride, polyvinylidene chloride,polytetrafluorethylene, polyethylene, polyamides, plastic, filter paper,dextran, germanium, silicon, (poly)tetrafluorethylene, gallium arsenide,gallium phosphide, silicon oxide, silicon nitrate and combinationsthereof. Methods of attaching a capture element as defined herein arewell known in the art and are defined hereinbelow. Additional solidsupports are also discussed hereinbelow.

[0061] As used herein, “affinity pair” refers to a pair of moieties (forexample complementary nucleic acid sequences, protein-ligand,antibody-antigen, protein subunits, and nucleic acid bindingproteins-binding sites) that can reversibly associate as a result ofattractive forces that exist between the moieties. An “affinity pair”includes the combination of a binding moiety and the correspondingcapture element and the combination of a tag and the correspondingcapture element.

[0062] In embodiments wherein the affinity pair comprises complementarynucleic acid regions that reversibly interact with one another, thelengths of the target nucleic acid binding sequences, and the nucleicacid sequences comprising the affinity pair, are chosen for the properthermodynamic functioning of the probe under the conditions of theprojected hybridization assay. Persons skilled in hybridization assayswill understand that pertinent conditions include probe, target andsolute concentrations, detection temperature, the presence ofdenaturants and volume excluders, and other hybridization-influencingfactors. The length of a target nucleic acid binding sequence can rangefrom 7 to about 10,000 nucleotides, preferably from 8-5000, 9-500, 9-250and most preferably, 10 to 140 nucleotides. If the probe is also anallele-discriminating probe, the length is more restricted, as isdiscussed below (this sentence has jumped in logic from a bindingmoiety:capture element concept to a probe:target concept).

[0063] In embodiments wherein the affinity pair comprises complementarynucleic acid regions that reversibly interact with one another, andcannot hybridize or are not complementary to a target nucleic acid, thecomplementary nucleic acid region sequences of the affinity pair shouldbe of sufficient length that under the conditions of the assay and atthe detection temperature, when the probe is not bound to a target, thestructure of the probe is such that the binding moiety of the probe willnot bind to the capture element, e.g., the complementary nucleic acidsequences are associated. Depending upon the assay conditions used,complementary nucleic acid sequences of 3-25 nucleotide lengths canperform this function. An intermediate range of 4-15, and morepreferably 5-11 , nucleotides is often appropriate. The actual lengthwill be chosen with reference to the target nucleic acid bindingsequence such that the secondary structure of the probe is stable whennot bound to the target nucleic acid at the temperature at whichcleavage of a cleavage structure comprising the probe bound to a targetnucleic acid is performed. As the target nucleic acid binding sequenceincreases in size up to 100 nucleotides, the length of the complementarynucleic acid sequences may increase up to 15-25 nucleotides. For atarget nucleic acid binding sequence greater than 100 nucleotides, thelength of the complementary nucleic acid sequences need not be increasedfurther. If the probe is also an allele-discriminating probe, thelengths of the complementary nucleic acid sequences are more restricted,as is discussed below.

[0064] Allele-discriminating probes that do not hybridize sufficientlyto a target-like nucleic acid sequence that contains one or morenucleotide mismatches as compared to the target nucleic acidcomplementary sequence, must be designed such that, under the assayconditions used, reduction or elimination of secondary structure in theprobe and hybridization with a target nucleic acid will occurefficiently only when the target nucleic acid complementary sequencefinds a perfectly complementary target sequence under certain reactionconditions. Certain reaction conditions may include, for example, aparticular temperature or a range of temperatures determined byexperimental optimization to permit an allele discriminating probe todiscriminate between a target and a target-like sequence with at least asingle nucleotide difference.

[0065] In one embodiment, an “allele-discriminating probe” according tothe invention refers to a probe that hybridizes to a target-like nucleicacid sequence that varies by at least one nucleotide from the targetnucleic acid, wherein the variant nucleotide(s) is/are not located inthe allele-discriminating site. According to this embodiment of theinvention, “an allele-discriminating probe” cannot bind efficiently to atarget-like nucleic acid sequence that also varies by at least onenucleotide in the allele-discriminating site under certain reactionconditions. Certain reaction conditions may include, for example, aparticular temperature or a range of temperatures determined byexperimental optimization to permit an allele discriminating probe todiscriminate between a target and a target-like sequence with at least asingle nucleotide difference.

[0066] In one embodiment of the invention, an allele discriminatingprobe according to the invention preferably comprises a target nucleicacid binding sequence from 6 to 50 and preferably from 7 to 25nucleotides, and complementary nucleic acid sequences from 3 to 8nucleotides. The guanosine-cytidine content of the secondary structureand probe-target hybrids, salt, and assay temperature should all beconsidered, for example magnesium salts have a strong stabilizing effectthat is particularly important to consider when designing short,allele-discriminating probes.

[0067] If an allele-discriminating probe is to have a target nucleicacid binding sequence of about 50 nucleotides long, the sequence shouldbe designed such that a single nucleotide mismatch to be discriminatedagainst occurs at or near the middle of the target nucleic acidcomplementary sequence. For example, probes comprising a sequence thatis 21 nucleotides long should preferably be designed so that themismatch occurs opposite one of the 14 most centrally locatednucleotides of the target nucleic acid complementary sequence and mostpreferably opposite one of the 7 most centrally located nucleotides.Designing a probe so that the mismatch to be discriminated againstoccurs in or near the middle of the target nucleic acid bindingsequence/target-like nucleic acid binding sequence is believed toimprove the performance of an allele-discriminating probe.

[0068] As used herein a “nuclease” or a “cleavage agent” refers to anenzyme that is specific for, that is, cleaves a cleavage structureaccording to the invention and is not specific for, that is, does notsubstantially cleave either a probe or a primer that is not hybridizedto a target nucleic acid, or a target nucleic acid that is nothybridized to a probe or a primer. The term “nuclease” includes anenzyme that possesses 5′ endonucleolytic activity for example a DNApolymerase, e.g. DNA polymerase I from E. coli, and DNA polymerase fromThermus aquaticus (Taq), Thermus thermophilus (Tth), and Thermus flavus(Tfl). The term nuclease also embodies FEN nucleases. The term “FENnuclease” encompasses an enzyme that possesses 5′ exonuclease and/or anendonuclease activity. The term “FEN nuclease” also embodies a 5′flap-specific nuclease. A nuclease or cleavage agent according to theinvention includes but is not limited to a FEN nuclease enzyme derivedfrom Archaeglobus fulgidus, Methanococcus jannaschii, Pyrococcusfuriosus, human, mouse or Xenopus laevis. A nuclease according to theinvention also includes Saccharomyces cerevisiae RAD27, andSchizosaccharomyces pombe RAD2, Pol I DNA polymerase associated 5′ to 3′exonuclease domain, (e.g. E. coli, Thermus aquaticus (Taq), Thermusflavus (Tfl), Bacillus caldotenax (Bca), Streptococcus pneumoniae) andphage functional homologs of FEN including but not limited to T5 5′ to3′ exonuclease, T7 gene 6 exonuclease and T3 gene 6 exonuclease.Preferably, only the 5′ to 3′ exonuclease domains of Taq, Tfl and BcaFEN nuclease are used. The term “nuclease” does not include RNAse H.

[0069] As used herein, “captured” as it refers to capture of a bindingmoiety by a capture element or capture of a tag by a capture element,means specifically bound by hydrogen bonding, covalent bonding, or viaan interaction between, for example a protein and a ligand, an antibodyand an antigen, protein subunits, a nucleic acid binding protein and anucleic acid binding site, or between complementary nucleic acids,wherein one member of the interacting pair is attached to a solidsupport. Under conditions of stable capture, binding results in theformation of a heterodimer with a dissociation constant (KD) of at leastabout 1×10³ M⁻¹, usually at least 1×10⁴ M⁻¹, typically at least 1×10⁵M⁻¹, preferably at least 1×10⁶ M⁻¹ to 1×10⁷ M⁻¹ or more, under suitableconditions. Methods of performing binding reactions between a captureelement, as defined herein, and a binding moiety or tag, as definedherein, are well-known in the art and are described hereinbelow. Methodsof attaching a capture element according to the invention to a solidsupport, as defined herein, are well known in the art and are definedhereinbelow.

[0070] As used herein, “wild type” refers to a gene or gene productwhich has the characteristics of (i.e., either has the sequence of orencodes, for the gene, or possesses the sequence or activity of, for anenzyme) that gene or gene product when isolated from a naturallyoccurring source.

[0071] A “5′ flap-specific nuclease” (also referred to herein as a“flap-specific nuclease”) according to the invention is an endonucleasewhich can remove a single stranded flap that protrudes as a 5′ singlestrand. In one embodiment of the invention, a flap-specific nucleaseaccording to the invention can also cleave a pseudo-Y structure. Asubstrate of a flap-specific nuclease according to the invention,comprises a target nucleic acid and an oligonucleotide probe, as definedherein, that comprises a region or regions that are complementary to thetarget nucleic acid. In another embodiment, a substrate of aflap-specific nuclease according to the invention comprises a targetnucleic acid, an upstream oligonucleotide that is complementary to thetarget nucleic acid and a downstream probe, according to the invention,that comprises a region or regions that are complementary to the targetnucleic acid. In one embodiment, the upstream oligonucleotide and thedownstream probe hybridize to non-overlapping regions of the targetnucleic acid. In another embodiment the upstream oligonucleotide and thedownstream probe hybridize to adjacent regions of the target nucleicacid.

[0072] As used herein, “adjacent” refers to separated by less than 20nucleotides, e.g., 15 nucleotides, 10 nucleotides, 5 nucleotides, or 0nucleotides.

[0073] A substrate of a flap-specific nuclease according to theinvention, also comprises a target nucleic acid, a second nucleic acid,a portion of which specifically hybridizes with a target nucleic acid,and a primer extension product from a third nucleic acid thatspecifically hybridizes with a target nucleic acid.

[0074] As used herein, a “cleavage structure” refers to a polynucleotidestructure (for example as illustrated in FIG. 1) comprising at least aduplex nucleic acid having a single stranded region comprising a flap, aloop, a single-stranded bubble, a D-loop, a nick or a gap. A cleavagestructure according to the invention thus includes a polynucleotidestructure comprising a flap strand of a branched DNA wherein a 5′single-stranded polynucleotide flap extends from a position near itsjunction to the double stranded portion of the structure and preferablythe flap is labeled with a detectable label. A flap of a cleavagestructure according to the invention is preferably about 1-10,000nucleotides, more preferably about 5-25 nucleotides and most preferablyabout 10-20 nucleotides and is preferably cleaved at a position locatedat the phosphate positioned at the “elbow” of the branched structure orat any of one to ten phosphates located proximal and/or distal from theelbow of the flap strand. As used herein, “elbow” refers to thephosphate bond between the first single stranded nucleotide of the 5′flap and the first double stranded (e.g., hybridized to the targetnucleic acid) nucleotide. In one embodiment, a flap of a cleavagestructure cannot hybridize to a target nucleic acid.

[0075] A cleavage structure according to one embodiment of the inventionpreferably comprises a target nucleic acid, and also may include anoligonucleotide probe according to the invention, that specificallyhybridizes with the target nucleic acid via a region or regions that arecomplementary to the target nucleic acid, and a flap extending from thehybridizing oligonucleotide probe. In another embodiment of theinvention, a cleavage structure comprises a target nucleic acid (forexample B in FIG. 4), an upstream oligonucleotide that is complementaryto the target sequence (for example A in FIG. 4), and a downstreamoligonucleotide probe according to the invention and comprising a regionor regions, that are complementary to the target sequence (for example Cin FIG. 4). In one embodiment, the upstream oligonucleotide and thedownstream probe hybridize to non-overlapping regions of the targetnucleic acid. In another embodiment, the upstream oligonucleotide andthe downstream probe hybridize to adjacent regions of the target nucleicacid.

[0076] A cleavage structure according to the invention may be apolynucleotide structure comprising a flap extending from the downstreamoligonucleotide probe of the invention, wherein the flap is formed byextension of the upstream oligonucleotide by the synthetic activity of anucleic acid polymerase, and subsequent, partial, displacement of the 5′end of the downstream oligonucleotide. In such a cleavage structure, thedownstream oligonucleotide may be blocked at the 3′ terminus to preventextension of the 3′ end of the downstream oligonucleotide.

[0077] A cleavage structure according to one embodiment of the inventionmay be formed by hybridizing a target nucleic acid with anoligonucleotide probe wherein the oligonucleotide probe has a secondarystructure that changes upon binding of the probe to the target nucleicacid, and further comprises a binding moiety and a complementary regionthat anneals to the target nucleic acid, and a non-complementary regionthat does not anneal to the target nucleic acid and forms a 5′ flap.

[0078] A cleavage structure also may be a pseudo-Y structure wherein apseudo Y-structure is formed if the strand upstream of a flap (referredto herein as a flap adjacent strand or primer strand) is not present,and double stranded DNA substrates containing a gap or nick. A “cleavagestructure”, as used herein, does not include a double stranded nucleicacid structure with only a 3′ single-stranded flap. As used herein, a“cleavage structure” comprises ribonucleotides or deoxyribonucleotidesand thus can be RNA or DNA.

[0079] A cleavage structure according to the invention may be anoverlapping flap wherein the 3′ end of an upstream oligonucleotidecapable of hybridizing to a target nucleic acid (for example A in FIG.4) is identical to 1 base pair of the downstream oligonucleotide probeof the invention (for example C in FIG. 4) that is annealed to a targetnucleic acid and wherein the overlap is directly downstream of the pointof extension of the single stranded flap.

[0080] A cleavage structure according to one embodiment of the inventionis formed by the steps of 1. incubating a) an upstream 3′ end,preferably an oligonucleotide primer, b) an oligonucleotide probelocated not more than 10,000 nucleotides downstream of the upstreamprimer and having a secondary structure that changes upon binding of theprobe to the target nucleic acid and further comprising a binding moietyc) an appropriate target nucleic acid wherein the target sequence is atleast partially complementary to both the upstream primer and downstreamprobe and d) a suitable buffer, under conditions that allow the nucleicacid sequence to hybridize to the oligonucleotide primers, and, in oneembodiment of the invention, 2. extending the 3′ end of the upstreamoligonucleotide primer by the synthetic activity of a polymerase suchthat the newly synthesized 3′ end of the upstream oligonucleotide primerbecomes adjacent to and/or displaces at least a portion of (i.e., atleast 1-10 nucleotides of) the 5′ end of the downstream oligonucleotideprobe. According to the method of the invention, buffers and extensiontemperatures are favorable for strand displacement by a particularnucleic acid polymerase according to the invention. Preferably, thedownstream oligonucleotide is blocked at the 3′ terminus to preventextension of the 3′ end of the downstream oligonucleotide.

[0081] In another embodiment of the invention, a cleavage structureaccording to the invention can be prepared by incubating a targetnucleic acid with an oligonucleotide probe having a secondary structurethat changes upon binding of the probe to the target nucleic acid, andfurther comprising a binding moiety and a non-complementary 5′ regionthat does not anneal to the target nucleic acid and forms a 5′ flap, anda complementary 3′ region that anneals to the target nucleic acid.

[0082] In another embodiment of the invention, a cleavage structureaccording to the invention can be prepared by incubating a targetnucleic acid with a downstream oligonucleotide probe having a secondarystructure that changes upon binding of the probe to the target nucleicacid, and further comprising a binding moiety and a non-complementary 5′region that does not anneal to the target nucleic acid and forms a 5′flap and a complementary 3′ region that anneals to the target nucleicacid, and an upstream oligonucleotide primer. In one embodiment, theupstream oligonucleotide and the downstream probe hybridize tonon-overlapping regions of the target nucleic acid. In anotherembodiment, the upstream oligonucleotide and the downstream probehybridize to adjacent regions of the target nucleic acid.

[0083] In a preferred embodiment of the invention a cleavage structureis labeled. A labeled cleavage structure according to one embodiment ofthe invention is formed by the steps of 1. incubating a) an upstreamextendable 3′ end, for example, an oligonucleotide primer, b) a labeledprobe having a secondary structure that changes upon binding of theprobe to the target nucleic acid, and further comprising a bindingmoiety, preferably located not more than 10,000 and more preferablylocated not more than 500 nucleotides downstream of the upstream primerand c) an appropriate target nucleic acid wherein the target sequence iscomplementary to both the primer and the labeled probe and d) a suitablebuffer, under conditions that allow the nucleic acid sequence tohybridize to the primers, and, in one embodiment of the invention, 2.extending the 3′ end of the upstream primer by the synthetic activity ofa polymerase such that the newly synthesized 3′ end of the upstreamprimer partially displaces the 5′ end of the downstream probe. Accordingto the method of the invention, buffers and extension temperatures arefavorable for strand displacement by a particular nucleic acidpolymerase according to the invention. Preferably, the downstreamoligonucleotide is blocked at the 3′ terminus to prevent extension ofthe 3′ end of the downstream oligonucleotide. In one embodiment, theupstream primer and the downstream probe hybridize to non-overlappingregions of the target nucleic acid.

[0084] In another embodiment, a cleavage structure according to theinvention can be prepared by incubating a target nucleic acid with aprobe having a secondary structure that changes upon binding of theprobe to the target nucleic acid, and further comprising a bindingmoiety and a non-complementary, labeled, 5′ region that does not annealto the target nucleic acid and forms a 5′ flap, and a complementary 3′region that anneals to the target nucleic acid. In another embodiment, acleavage structure according to the invention can be prepared byincubating a target nucleic acid with a downstream probe having asecondary structure that changes upon binding of the probe to the targetnucleic acid, and further comprising a binding moiety and anon-complementary, labeled, 5′ region that does not anneal to the targetnucleic acid and forms a 5′ flap and a complementary 3′ region thatanneals to the target nucleic acid, and an upstream oligonucleotideprimer. In one embodiment, the upstream oligonucleotide and thedownstream probe hybridize to non-overlapping regions of the targetnucleic acid. In another embodiment, the upstream oligonucleotide andthe downstream probe hybridize to adjacent regions of the target nucleicacid.

[0085] As used herein, “generating a signal” refers to detecting and ormeasuring a released nucleic acid fragment that is released from thecleavage structure and is captured by binding of a binding moiety to acapture element on a solid support, as an indication of the presence ofa target nucleic acid in a sample.

[0086] As used herein, “sample” refers to any substance containing orpresumed to contain a nucleic acid of interest (a target nucleic acid)or which is itself a nucleic acid containing or presumed to contain atarget nucleic acid of interest. The term “sample” thus includes asample of nucleic acid (genomic DNA, cDNA, RNA), cell, organism, tissue,fluid, or substance including but not limited to, for example, plasma,serum, spinal fluid, lymph fluid, synovial fluid, urine, tears, stool,external secretions of the skin, respiratory, intestinal andgenitourinary tracts, saliva, blood cells, tumors, organs, tissue,samples of in vitro cell culture constituents, natural isolates (such asdrinking water, seawater, solid materials), microbial specimens, andobjects or specimens that have been “marked” with nucleic acid tracermolecules.

[0087] As used herein, “target nucleic acid” or “template nucleic acidsequence” refers to a region of a nucleic acid that is to be eitherreplicated, amplified, and/or detected. In one embodiment, the “targetnucleic acid” or “template nucleic acid sequence” resides between twoprimer sequences used for amplification.

[0088] As used herein, “nucleic acid polymerase” refers to an enzymethat catalyzes the polymerization of nucleoside triphosphates.Generally, the enzyme will initiate synthesis at the 3′-end of theprimer annealed to the target sequence, and will proceed in the5′-direction along the template, and if possessing a 5′ to 3′ nucleaseactivity, hydrolyzing intervening, annealed probe to release bothlabeled and unlabeled probe fragments, until synthesis terminates. KnownDNA polymerases include, for example, E. coli DNA polymerase I, T7 DNApolymerase, Thermus thermophilus (Tth) DNA polymerase, Bacillusstearothermophilus DNA polymerase, Thermococcus litoralis DNApolymerase, Thermus aquaticus (Taq) DNA polymerase and Pyrococcusfuriosus (Pfu) DNA polymerase.

[0089] As used herein, “5′ to 3′ exonuclease activity” or “5′→3′exonuclease activity” refers to that activity of a template-specificnucleic acid polymerase e.g. a 5′→3′ exonuclease activity traditionallyassociated with some DNA polymerases whereby mononucleotides oroligonucleotides are removed from the 5′ end of a polynucleotide in asequential manner, (i.e., E. coli DNA polymerase I has this activitywhereas the Klenow (Klenow et al., 1970, Proc. Natl. Acad. Sci., USA,65:168) fragment does not, (Klenow et al., 1971, Eur. J. Biochem.,22:371)), or polynucleotides are removed from the 5′ end by anendonucleolytic activity that may be inherently present in a 5′ to 3′exonuclease activity.

[0090] As used herein, the phrase “substantially lacks 5′ to 3′exonuclease activity” or “substantially lacks 5′→3′ exonucleaseactivity” means having less than 10%, 5%, 1%, 0.5%, or 0.1% of theactivity of a wild type enzyme. The phrase “lacking 5′ to 3′ exonucleaseactivity” or “lacking 5′→3′ exonuclease activity” means havingundetectable 5′ to 3′ exonuclease activity or having less than about 1%,0.5%, or 0.1% of the 5′to 3′exonuclease activity of a wild type enzyme.

[0091] To detect structure-specific endonucleolytic activity, a DNAtemplate consisting of a flap structure, wherein the downstream flapoligonucleotide is radiolabeled at the 5′ end is employed. The reactionis carried out with DNA polymerase in the presence of dNTPs (to extendthe upstream primer). Radiolabeled cleavage products are visualized bygel electrophoresis (Lyamichev et al., 1993, Science 260: 778).

[0092] Alternatively, the 5′-3′ exonuclease activity of a DNA polymeraseis assayed using uniformly-labeled double-stranded DNA that is alsonicked. The release of radioactivity (TCA soluble cpms) by a DNApolymerase in the absence and presence of dNTPs is measured.Non-proofreading DNA polymerases with 5′-3′ exonuclease activity arestimulated 10-fold or more by concomitant polymerization that occurs inthe presence of dNTPs (increase in cpms released in the presence ofdNTPs). Proofreading DNA polymerases with 3′-5′ exo activity areinhibited completely by concomitant polymerization that occurs in thepresence of dNTPs (decrease in cpms released in the presence of dNTPs)(U.S. Pat. No. 5,352,778).

[0093] Nucleases useful according to the invention include any enzymethat possesses 5′ endonucleolytic activity for example a DNA polymerase,e.g. DNA polymerase I from E. coli, and DNA polymerase from Thermusaquaticus (Taq), Thermus thermophilus (Tth), and Thermus flavus (Tfl).Nucleases useful according to the invention also include DNA polymeraseswith 5′-3′ exonuclease activity, including but not limited toeubacterial DNA polymerase I, including enzymes derived from Thermusspecies (Taq, Tfl, Tth, Tca (caldophilus) Tbr (brockianus)), enzymesderived from Bacillus species (Bst, Bca, Magenta (full lengthpolymerases, NOT N-truncated versions)), enzymes derived from Thermotogaspecies (Tma (maritima, Tne (neopolitana)) and E. coli DNA polymerase I.The term nuclease also embodies FEN nucleases. Additional nucleic acidpolymerases useful according to the invention are included below in thesection entitled, “Nucleic Acid Polymerases”.

[0094] As used herein, “cleaving” refers to enzymatically separating acleavage structure into distinct (i.e. not physically linked to otherfragments or nucleic acids by phosphodiester bonds) fragments ornucleotides and fragments that are released from the cleavage structure.For example, cleaving a labeled cleavage structure refers to separatinga labeled cleavage structure according to the invention and definedbelow, into distinct fragments including fragments derived from anoligonucleotide that specifically hybridizes with a target nucleic acidor wherein one of the distinct fragments is a labeled nucleic acidfragment derived from a target nucleic acid and/or derived from anoligonucleotide that specifically hybridizes with a target nucleic acidthat can be detected and/or measured by methods well known in the artand described herein that are suitable for detecting the labeled moietythat is present on a labeled fragment.

[0095] As used herein, “endonuclease” refers to an enzyme that cleavesbonds, preferably phosphodiester bonds, within a nucleic acid molecule.An endonuclease according to the invention can be specific forsingle-stranded or double-stranded DNA or RNA.

[0096] As used herein, “exonuclease” refers to an enzyme that cleavesbonds, preferably phosphodiester bonds, between nucleotides one at atime from the end of a polynucleotide. An exonuclease according to theinvention can be specific for the 5′ or 3′ end of a DNA or RNA molecule,and is referred to herein as a 5′ exonuclease or a 3′ exonuclease.

[0097] As used herein a “flap” refers to a region of single stranded DNAthat extends from a double stranded nucleic acid molecule. A flapaccording to the invention is preferably between about 1-10,000nucleotides, more preferably between about 5-25 nucleotides and mostpreferably between about 10-20 nucleotides.

[0098] In a preferred embodiment, the binding moiety is a tag.

[0099] In another preferred embodiment, the binding moiety is a nucleicacid sequence that binds to a capture element.

[0100] The invention also provides a method of detecting or measuring atarget nucleic acid comprising the steps of: forming a cleavagestructure by incubating a sample containing a target nucleic acid with aprobe having a secondary structure that changes upon binding of theprobe to the target nucleic acid and, the probe further comprising abinding moiety, cleaving the cleavage structure with a nuclease torelease a nucleic acid fragment wherein the cleavage is performed at acleaving temperature, and the secondary structure of the probe when notbound to the target nucleic acid is stable at or below the cleavingtemperature; and detecting and/or measuring the amount of the fragmentcaptured by binding of the binding moiety to a capture element on asolid support as an indication of the presence of the target sequence inthe sample.

[0101] As used herein, “detecting a target nucleic acid” or “measuring atarget nucleic acid” refers to determining the presence of a particulartarget nucleic acid in a sample or determining the amount of aparticular target nucleic acid in a sample as an indication of thepresence of a target nucleic acid in a sample. The amount of a targetnucleic acid that can be measured or detected is preferably about 1molecule to 10²⁰ molecules, more preferably about 100 molecules to 10¹⁷molecules and most preferably about 1000 molecules to 10¹⁴ molecules.According to one embodiment of the invention, the detected nucleic acidis derived from the labeled 5′ end of a downstream probe of a cleavagestructure according to the invention (for example C in FIG. 4), that isdisplaced from the target nucleic acid by the 3′ extension of anupstream probe of a cleavage structure according to the invention (forexample A of FIG. 4). According to the present invention, a label isattached to the 5′ end of the downstream probe (for example C in FIG. 4)comprising a cleavage structure according to the invention.Alternatively, a label is attached to the 3′ end of the downstream probeand a quencher is attached to the 5′ flap of the downstream probe.According to the invention, a label may be attached to the 3′ end of thedownstream probe (for example C in FIG. 4) comprising a cleavagestructure according to the invention.

[0102] According to the invention, the downstream probe (for example Cin FIG. 4) may be labeled internally. In a preferred embodiment, acleavage structure according to the invention can be prepared byincubating a target nucleic acid with a probe having a secondarystructure that changes upon binding of the probe to the target nucleicacid, and further comprising a non-complementary, labeled, 5′ regionthat does not anneal to the target nucleic acid and forms a 5′ flap, anda complementary 3′ region that anneals to the target nucleic acid.According to this embodiment of the invention, the detected nucleic acidis derived from the labeled 5′ flap region of the probe. Preferablythere is a direct correlation between the amount of the target nucleicacid and the signal generated by the cleaved, detected nucleic acid.

[0103] In another embodiment, the probe is labeled with a pair ofinteractive labels (e.g., a FRET or non-FRET pair) positioned to permitthe separation of the labels during oligonucleotide probe unfolding(e.g., for example due to a change in the secondary structure of theprobe) or hydrolysis. As used herein, “detecting the amount of thefragment captured by a capture element on a solid support” or “measuringthe amount of the fragment captured by a capture element on a solidsupport” or “detecting the amount of the fragment captured by a captureelement on a solid support” or “measuring the amount of the fragmentcaptured by a capture element on a solid support” refers to determiningthe presence of a labeled or unlabeled fragment in a sample ordetermining the amount of a labeled or unlabeled fragment in a sample.Methods well known in the art and described herein can be used to detector measure release of labeled or unlabeled fragments bound to a captureelement on a solid support, or following the release of the labeled orunlabeled fragment from a capture element on a solid support. Thedetection methods described herein are operative for detecting afragment wherein any amount of a fragment is detected whether that be asmall or large proportion of the fragments generated in the reaction. Amethod of detecting or measuring release of labeled fragments will beappropriate for measuring or detecting the labeled moiety that ispresent on the labeled fragments bound to a capture element on a solidsupport. Methods of detecting or measuring release of unlabeledfragments include, for example, gel electrophoresis or by hybridization,according to methods well known in the art. The detection methodsdescribed herein are operative when as little as 1 or 2 molecules (andup to 1 or 2 million, for example 10, 100, 1000, 10,000, 1 million) ofreleased fragment are detected.

[0104] As used herein, “labeled fragments” refer to cleavedmononucleotides or small oligonucleotides or oligonucleotides derivedfrom the labeled cleavage structure according to the invention whereinthe cleaved oligonucleotides are preferably between about 1-1000nucleotides, more preferably between about 5-50 nucleotides and mostpreferably between about 16-18 nucleotides, which are cleaved from acleavage structure by a nuclease and can be detected by methods wellknown in the art and described herein.

[0105] In one embodiment, a probe is a bi-molecular or multimolecularprobe wherein a first molecule comprising the probe is labeled with afluorophore and a second molecule comprising the probe is labeled with aquencher. As used herein, a “subprobe” and “subquencher” refer to afirst molecule of a bi- or multi-molecular probe according to theinvention, that is labeled with a fluorophore and a second molecule of abi- or multi-molecular probe according to the invention, that is labeledwith a quencher, respectively. According to this embodiment, followingbinding of the bi- or multi-molecular probe to the target nucleic acid,and cleavage by a nuclease, the subprobe and subquencher dissociate fromeach other (that is, the distance between the subprobe and thesubquencher increases) and a signal is generated as a result of thisdissociation and subsequent separation of the subprobe and subquencher.

[0106] In a preferred embodiment, the binding moiety is a tag.

[0107] In another preferred embodiment, the binding moiety is a nucleicacid sequence that binds to a capture element.

[0108] In a preferred embodiment, the method further comprises a nucleicacid polymerase.

[0109] In another preferred embodiment, the cleavage structure furthercomprises a 5′ flap.

[0110] In another preferred embodiment, the cleavage structure furthercomprises an oligonucleotide primer.

[0111] In another preferred embodiment, the secondary structure isselected from the group consisting a stem-loop structure, a hairpinstructure, an internal loop, a bulge loop, a branched structure, apseudoknot structure or a cloverleaf structure.

[0112] In another preferred embodiment, the nuclease is a FEN nuclease.

[0113] In another preferred embodiment the FEN nuclease is selected fromthe group consisting of FEN nuclease enzyme derived from Archaeglobusfulgidus, Methanococcus jannaschii, Pyrococcus furiosus, human, mouse orXenopus laevis. A FEN nuclease according to the invention also includesSaccharomyces cerevisiae RAD27, and Schizosaccharomyces pombe RAD2, PolI DNA polymerase associated 5′ to 3′ exonuclease domain, (e.g. E. coli,Thermus aquaticus (Taq), Thermus flavus (Tfl), Bacillus caldotenax(Bca), Streptococcus pneumoniae) and phage functional homologs of FENincluding but not limited to T4, T5 5′ to 3′ exonuclease, T7 gene 6exonuclease and T3 gene 6 exonuclease.

[0114] Preferably, only the 5′ to 3′ exonuclease domains of Taq, Tfl andBca FEN nuclease are used.

[0115] In another preferred embodiment, the probe further comprises areporter.

[0116] In another preferred embodiment, the reporter comprises a tag.

[0117] In another preferred embodiment, the fragment is captured bybinding of the tag to a capture element.

[0118] In another preferred embodiment, the cleavage structure is formedcomprising at least one labeled moiety capable of providing a signal.

[0119] In another preferred embodiment, the cleavage structure is formedcomprising a pair of interactive signal generating labeled moietieseffectively positioned on the probe to quench the generation of adetectable signal when the probe is not bound to the target nucleicacid.

[0120] In another preferred embodiment, the labeled moieties areseparated by a site susceptible to nuclease cleavage, thereby allowingthe nuclease activity of the nuclease to separate the first interactivesignal generating labeled moiety from the second interactive signalgenerating labeled moiety by cleaving at the site susceptible tonuclease cleavage, thereby generating a detectable signal.

[0121] The presence of a pair of interactive signal generating labeledmoieties, as described above, allows for discrimination betweenannealed, uncleaved probe that may bind to a capture element, andreleased labeled fragment that is bound to a capture element.

[0122] In another preferred embodiment, the pair of interactive signalgenerating moieties comprises a quencher moiety and a fluorescentmoiety.

[0123] The invention also provides for a polymerase chain reactionprocess for detecting a target nucleic acid in a sample. This processcomprises, providing a cleavage structure comprising a probe having asecondary structure that changes upon binding of the probe to the targetnucleic acid and, the probe further comprising a binding moiety, a setof oligonucleotide primers wherein a first primer contains a sequencecomplementary to a region in one strand of the target nucleic acid andprimes the synthesis of a complementary DNA strand, and a second primercontains a sequence complementary to a region in a second strand of thetarget nucleic acid and primes the synthesis of a complementary DNAstrand. This process also comprises amplifying the target nucleic acidemploying a nucleic acid polymerase as a template-dependent polymerizingagent under conditions which are permissive for PCR cycling steps of (i)annealing of primers required for amplification to a template nucleicacid sequence contained within the target nucleic acid, (ii) extendingthe primers providing that the nucleic acid polymerase synthesizes aprimer extension product, and (iii) cleaving the cleavage structureemploying a nuclease as a cleavage agent for release of labeledfragments from the cleavage structure thereby creating detectablelabeled fragments. According to this process, the cleaving is performedat a cleaving temperature and the secondary structure of the secondprimer when not bound to the target nucleic acid is stable at or belowthe cleaving temperature. The amount of released, labeled fragmentcaptured by binding of the binding moiety to a capture element on asolid support is detected and/or measured as an indicator of thepresence of the target sequence in the sample.

[0124] As used herein, an “oligonucleotide primer” refers to a singlestranded DNA or RNA molecule that is hybridizable to a nucleic acidtemplate and primes enzymatic synthesis of a second nucleic acid strand.Oligonucleotide primers useful according to the invention are betweenabout 6 to 100 nucleotides in length, preferably about 17-50 nucleotidesin length and more preferably about 17-45 nucleotides in length.Oligonucleotide probes useful for the formation of a cleavage structureaccording to the invention are between about 17-40 nucleotides inlength, preferably about 17-30 nucleotides in length and more preferablyabout 17-25 nucleotides in length.

[0125] As used herein, “template dependent polymerizing agent” refers toan enzyme capable of extending an oligonucleotide primer in the presenceof adequate amounts of the four deoxyribonucleoside triphosphates (dATP,dGTP, dCTP and dTTP) or analogs as described herein, in a reactionmedium comprising appropriate salts, metal cations, appropriatestabilizers and a pH buffering system. Template dependent polymerizingagents are enzymes known to catalyze primer- and template- dependent DNAsynthesis, and possess 5′ to 3′ nuclease activity. Preferably, atemplate dependent polymerizing agent according to the invention lacks5′ to 3′ nuclease activity.

[0126] As used herein, “amplifying” refers to producing additionalcopies of a nucleic acid sequence, including the method of thepolymerase chain reaction.

[0127] In a preferred embodiment, the nuclease is a FEN nuclease.

[0128] In a preferred embodiment, the binding moiety is a tag.

[0129] In another preferred embodiment, the binding moiety is a nucleicacid sequence that binds to a capture element.

[0130] In another preferred embodiment, the oligonucleotide primers ofstep b are oriented such that the forward primer is located upstream ofthe cleavage structure and the reverse primer is located downstream ofthe cleavage structure.

[0131] In another preferred embodiment, the nucleic acid polymerase hasstrand displacement activity.

[0132] Nucleic acid polymerases exhibiting strand displacement activityand useful according to the invention include but are not limited toarchaeal DNA polymerases with “temperature activated” stranddisplacement activity (exo plus and exo minus versions of Vent, DeepVent, Pfu, JDF-3, KOD (LTI's tradename Pfx), Pwo, 9 degrees North,Thermococcus aggregans, Thermococcus gorgonarius), and eubacterial DNApolymerases with strand displacement activity (exo minus Bst, exo minusBca, Genta, Klenow fragment, exo minus Klenow fragment exo minus T7 DNApolymerase (Sequenase).

[0133] In another preferred embodiment, the nucleic acid polymerase isthermostable

[0134] In another preferred embodiment, the nuclease is thermostable.

[0135] As used herein, “thermostable” refers to an enzyme which isstable and active at temperatures as great as preferably between about90-100° C. and more preferably between about 70-98° C. to heat ascompared, for example, to a non-thermostable form of an enzyme with asimilar activity. For example, a thermostable nucleic acid polymerase orFEN nuclease derived from thermophilic organisms such as P. furiosus, M.jannaschii, A. fulgidus or P. horikoshii are more stable and active atelevated temperatures as compared to a nucleic acid polymerase from E.coli or a mammalian FEN enzyme. A representative thermostable nucleicacid polymerase isolated from Thermus aquaticus (Taq) is described inU.S. Pat. No. 4,889,818 and a method for using it in conventional PCR isdescribed in Saiki et al., 1988, Science 239:487. Another representativethermostable nucleic acid polymerase isolated from P. Furiosus (Pfu) isdescribed in Lundberg et al., 1991, Gene, 108:1-6. Additionalrepresentative temperature stable polymerases include, e.g., polymerasesextracted from the thermophilic bacteria Thermus flavus, Thermus ruber,Thermus thermophilus, Bacillus stearothermophilus (which has a somewhatlower temperature optimum than the others listed), Thermus lacteus,Thermus rubens, Thermotoga maritima, or from thermophilic archaeaThermococcus litoralis, and Methanothermus fervidus.

[0136] Temperature stable polymerases and FEN nucleases are preferred ina thermocycling process wherein double stranded nucleic acids aredenatured by exposure to a high temperature (about 95° C.) during thePCR cycle.

[0137] In another preferred embodiment, the nuclease is a flap-specificnuclease.

[0138] In another preferred embodiment, the probe further comprises areporter.

[0139] In another preferred embodiment, the reporter comprises a tag.

[0140] In another preferred embodiment, the fragment is captured bybinding of said tag to a capture element.

[0141] In another preferred embodiment, the cleavage structure is formedcomprising at least one labeled moiety capable of providing a signal.

[0142] In another preferred embodiment, the cleavage structure is formedcomprising a pair of interactive signal generating labeled moietieseffectively positioned on the probe to quench the generation of adetectable signal when the probe is not bound to the target nucleicacid.

[0143] In another preferred embodiment, the labeled moieties areseparated by a site susceptible to nuclease cleavage, thereby allowingthe nuclease activity of the nuclease to separate the first interactivesignal generating labeled moiety from the second interactive signalgenerating labeled moiety by cleaving at the site susceptible tonuclease cleavage, thereby generating a detectable signal.

[0144] In another preferred embodiment, the pair of interactive signalgenerating moieties comprises a quencher moiety and a fluorescentmoiety.

[0145] In another preferred embodiment, the nucleic acid polymerase isselected from the group consisting of Taq polymerase and Pfu polymerase.

[0146] The invention provides for a polymerase chain reaction processwherein amplification and detection of a target nucleic acid occurconcurrently (i.e., real time detection). The invention also providesfor a polymerase chain reaction process wherein amplification of atarget nucleic acid occurs prior to detection of the target nucleic acid(i.e., end point detection).

[0147] The invention also provides for a polymerase chain reactionprocess for simultaneously forming a cleavage structure, amplifying atarget nucleic acid in a sample and cleaving the cleavage structure.This process comprises the step of: (a) providing an upstreamoligonucleotide primer complementary to a first region in one strand ofthe target nucleic acid, a downstream labeled probe complementary to asecond region in the same strand of the target nucleic acid, wherein thedownstream labeled probe is capable of forming a secondary structurethat changes upon binding of the probe to the target nucleic acid and,the probe further comprises a binding moiety, and a downstreamoligonucleotide primer complementary to a region in a second strand ofthe target nucleic acid. According to this step of the process, theupstream primer primes the synthesis of a complementary DNA strand, andthe downstream primer primes the synthesis of a complementary DNAstrand. This process also comprises the step of (b) detecting a nucleicacid which is produced and captured by binding of the binding moiety toa capture element on a solid support. The nucleic acid that is detectedis produced in a reaction comprising amplification and cleavage of thetarget nucleic acid wherein a nucleic acid polymerase is atemplate-dependent polymerizing agent under conditions which arepermissive for PCR cycling steps of (i) annealing of primers to a targetnucleic acid, (ii) extending the primers of step (a), providing that thenucleic acid polymerase synthesizes primer extension products, and theprimer extension product of the upstream primer of step (a) partiallydisplaces the downstream probe of step (a) to form a cleavage structure.The conditions are also permissive for (iii) cleaving the cleavagestructure employing a nuclease as a cleavage agent for release ofdetectable labeled fragments from the cleavage structure. The cleavingis performed at a cleaving temperature and the secondary structure ofthe probe when not bound to the target nucleic acid is stable at orbelow the cleaving temperature.

[0148] In a preferred embodiment, the cleavage structure furthercomprises a 5′ flap.

[0149] The invention also provides a method of forming a cleavagestructure comprising the steps of: (a) providing a target nucleic acid,(b) providing an upstream primer complementary to the target nucleicacid, (c) providing a downstream probe having a secondary structure thatchanges upon binding of the probe to a target nucleic acid and, theprobe further comprises a binding moiety; and (d) annealing the targetnucleic acid, the upstream primer and the downstream probe. The cleavagestructure can be cleaved with a nuclease at a cleaving temperature. Thesecondary structure of the probe when not bound to the target nucleicacid is stable at or below the cleaving temperature.

[0150] In a preferred embodiment, the cleavage structure comprises a 5′flap.

[0151] The invention also provides for a composition comprising a targetnucleic acid, a probe having a secondary structure that changes uponbinding of the probe to a target nucleic acid and, the probe furthercomprises a binding moiety, and a nuclease. The probe and the targetnucleic acid of this composition can bind to form a cleavage structurethat can be cleaved by the nuclease at a cleaving temperature. Thesecondary structure of the probe when not bound to the target nucleicacid is stable at or below the cleaving temperature.

[0152] In a preferred embodiment, the composition further comprises anoligonucleotide primer.

[0153] In another preferred embodiment, the probe and theoligonucleotide hybridize to non-overlapping regions of the targetnucleic acid.

[0154] The invention also provides for a kit for generating a signalindicative of the presence of a target nucleic acid in a sample,comprising a probe having a secondary structure that changes uponbinding of the probe to a target nucleic acid and, the probe furthercomprising a binding moiety, and a nuclease. The probe of this kit canbind to a target nucleic acid to form a cleavage structure that can becleaved by the nuclease at a cleaving temperature. The secondarystructure of the probe when not bound to the target nucleic acid isstable at or below the cleaving temperature.

[0155] In a preferred embodiment, the kit further comprises anoligonucleotide primer.

[0156] In another preferred embodiment, the nuclease is a FEN nuclease.

[0157] In another preferred embodiment, the probe comprises at least onelabeled moiety.

[0158] In another preferred embodiment, the probe comprises a pair ofinteractive signal generating labeled moieties effectively positioned toquench the generation of a detectable signal when the probe is not boundto the target nucleic acid.

[0159] In another preferred embodiment, the labeled moieties areseparated by a site susceptible to nuclease cleavage, thereby allowingthe nuclease activity of the nuclease to separate the first interactivesignal generating labeled moiety from the second interactive signalgenerating labeled moiety by cleaving at the site susceptible tonuclease cleavage, thereby generating a detectable signal.

[0160] In another preferred embodiment, the pair of interactive signalgenerating moieties comprises a quencher moiety and a fluorescentmoiety.

[0161] Further features and advantages of the invention are as follows.The claimed invention provides a method of generating a signal to detectand/or measure a target nucleic acid wherein the generation of a signalis an indication of the presence of a target nucleic acid in a sample.The method of the claimed invention does not require multiple steps. Theclaimed invention also provides a PCR based method for detecting and/ormeasuring a target nucleic acid comprising generating a signal as anindication of the presence of a target nucleic acid. The claimedinvention allows for simultaneous amplification and detection and/ormeasurement of a target nucleic acid. The claimed invention alsoprovides a PCR based method for detecting and/or measuring a targetnucleic acid comprising generating a signal in the absence of a nucleicacid polymerase that demonstrates 5′ to 3′ exonuclease activity.

[0162] Further features and advantages of the invention will become morefully apparent in the following description of the embodiments anddrawings thereof, and from the claims.

BRIEF DESCRIPTION OF DRAWINGS

[0163]FIG. 1 demonstrates FEN nuclease cleavage structures.

[0164]FIG. 2 demonstrates three templates (labeled 1, 2, and 3) that maybe used to detect FEN nuclease activity.

[0165]FIG. 3 demonstrates secondary structures.

[0166]FIG. 4 is a diagram illustrating a synthesis and cleavage reactionto generate a signal according to the invention.

[0167]FIG. 5 is a Sypro Orange stained polyacrylamide gel demonstratingCBP-tagged PFU FEN-1 protein.

[0168]FIG. 6 is an autoradiograph of a FEN-1 nuclease assay.

[0169]FIG. 7 is a representation of an open circle probe for rollingcircle amplification.

[0170]FIG. 8 is a representation of rolling circle amplification.

[0171]FIG. 9 is a representation of a safety pin probe.

[0172]FIG. 10 is a representation of a scorpion probe.

[0173]FIG. 11 is a representation of a sunrise/amplifluor probe

[0174]FIG. 12a is a graph demonstrating the difference in lightabsorbance of double-stranded versus single-stranded DNA.

[0175]FIG. 12b is a graph demonstrating DNA melting curves.

[0176]FIG. 12c is a graph demonstrating the effects of temperature onthe relative optical absorbance of DNA.

[0177]FIG. 12d is a graph demonstrating the effects of temperature onthe relative optical absorbance of DNA.

[0178]FIG. 12e is a graph demonstrating the effects of temperature onthe fluorescence of DNA labeled with a pair of interactive labels.

[0179]FIG. 12f is a graph demonstrating the effects of temperature onthe fluorescence of DNA labeled with a pair of interactive labels.

[0180]FIG. 12g is a graph demonstrating the effects of a target nucleicacid on the fluorescence of DNA labeled with a pair of interactivelabels.

DESCRIPTION

[0181] The invention provides for a method of generating a signal todetect the presence of a target nucleic acid in a sample wherein anucleic acid is treated with the combination of a probe having asecondary structure that changes upon binding of the probe to a targetnucleic acid and comprising a binding moiety and a nuclease. Theinvention also provides for a process for detecting or measuring anucleic acid that allows for concurrent amplification, cleavage anddetection of a target nucleic acid in a sample.

[0182] The practice of the present invention will employ, unlessotherwise indicated, conventional techniques of molecular biology,microbiology and recombinant DNA techniques, which are within the skillof the art. Such techniques are explained fully in the literature. See,e.g., Sambrook, Fritsch & Maniatis, 1989, Molecular Cloning: ALaboratory Manual, Second Edition; Oligonucleotide Synthesis (M. J.Gait, ed., 1984); Nucleic Acid Hybridization (B. D. Hames & S. J.Higgins, eds., 1984); A Practical Guide to Molecular Cloning (B. Perbal,1984); and a series, Methods in Enzymology (Academic Press, Inc.); ShortProtocols In Molecular Biology, (Ausubel et al., ed., 1995). Allpatents, patent applications, and publications mentioned herein, bothsupra and infra, are hereby incorporated by reference.

[0183] I. Nucleases

[0184] Nucleases useful according to the invention include any enzymethat possesses 5′ endonucleolytic activity for example a DNA polymerase,e.g. DNA polymerase I from E. coli, and DNA polymerase from Thermusaquaticus (Taq), Thermus thermophilus (Tth), and Thermus flavus (Tfl).Nucleases useful according to the invention also include DNA polymeraseswith 5′-3′ exonuclease activity, including but not limited toeubacterial DNA polymerase I, including enzymes derived from Thermusspecies (Taq, Tfl, Tth, Tca (caldophilus) Tbr (brockianus)),enzymesderived from Bacillus species (Bst, Bca, Magenta (full lengthpolymerases, NOT N-truncated versions)), enzymes derived from Thermotogaspecies (Tma (maritima, Tne (neopolitana)) and E. coli DNA polymerase I.The term nuclease also embodies FEN nucleases. A nuclease usefulaccording to the invention cannot cleave either a probe or primer thatis not hybridized to a target nucleic acid or a target nucleic acid thatis not hybridized to a probe or a primer.

[0185] FEN-1 is an ˜40 kDa divalent metal ion-dependent exo- andendonuclease that specifically recognizes the backbone of a 5′single-stranded flap strand and tracks down this arm to the cleavagesite, which is located at the junction wherein the two strands of duplexDNA adjoin the single-stranded arm. Both the endo- and exonucleolyticactivities show little sensitivity to the base at the most 5′ positionat the flap or nick. Both FEN-1 endo- and exonucleolytic substratebinding and cutting are stimulated by an upstream oligonucleotide (flapadjacent strand or primer). This is also the case for E. coli pol I. Theendonuclease activity of the enzyme is independent of the 5′ flaplength, cleaving a 5′ flap as small as one nucleotide. The endonucleaseand exonuclease activities are insensitive to the chemical nature of thesubstrate, cleaving both DNA and RNA.

[0186] Both the endo- and exonucleolytic activities are inhibited byconcentrations of salts in the physiological range. The exonucleaseactivity is inhibited 50-fold at 50 mM NaCl as compared to 0 mM NaCl.The endonuclease activity is inhibited only sevenfold at 50 mM NaCl(Reviewed in Lieber 1997, supra).

[0187] Although a 5′-OH terminus is a good substrate for FEN-1 loadingonto a 5′ flap substrate, it serves as a very poor substrate when partof a nick in an otherwise double stranded DNA structure. Theelectrostatic repulsion by the terminal phosphate is likely to favorbreathing of the substrate into a pseudo-flap configuration, providingthe active form of the substrate for FEN-1. Such an explanation wouldindicate a single active site and a single mechanism of loading of FEN-1onto the 5′ ssDNA terminus of the flap or pseudo-flap configuration ofthe nick. Consistent with this model are observations that optimalactivity at a nick requires very low Mg²⁺ and monovalent saltconcentrations, which destabilize base-pairing and would favor breathingof a nick to a flap. Higher Mg²⁺ and monovalent salt concentrationswould disfavor breathing and inhibit cutting of nicked or gappedstructures that do require breathing to convert to a flap. Cleavage ofstable flap structures is optimal at moderate Mg²⁺ levels and does notdecrease with increasing Mg²⁺ concentration. This is because a flapsubstrate does not have to melt out base pairs to achieve its structure;hence, it is entirely insensitive to Mg²⁺. Though the endonucleolyticactivity decreases with monovalent salt, the decline is not nearly assharp as that seen for the exonucleolytic activity. Furthermore, it haspreviously been shown that one-nucleotide flaps are efficientsubstrates. All of these observations are consistent with the fact thatwhen FEN-1 has been interpreted to be functioning as an exonuclease, thesize of the degradation products vary from one to several nucleotides inlength. Breathing of nicks into flaps of varying length would beexpected to vary with local sequence, depending on the G/C content. Insummary, a nick breathing to form a transient flap means that theexonucleolytic activity of FEN-1 is the same as the endonucleolyticactivity (Reviewed in Lieber, 1997, supra).

[0188] The endonuclease and exonuclease activities of FEN-1 cleave bothDNA and RNA without requiring accessory proteins. At the replicationfork, however, FEN-1 does interact with other proteins, including a DNAhelicase and the proliferating cell nuclear antigen (PCNA), theprocessivity factor for DNA polymerases δ and ε. PCNA significantlystimulates FEN-1 endo- and exonucleolytic activity.

[0189] The FEN-1 enzymes are functionally related to several smallerbacteriophage 5′→3′ exonucleases such as T5 5′ exonuclease and T4 RNaseH as well as to the larger eukaryotic nucleotide excision repair enzymessuch as XPG, which also acts in the transcription-coupled repair ofoxidative base damage. In eubacteria such as Escherichia coli andThermus aquaticus, Okazaki processing is provided by the PolI 5′→3′exonuclease domain. These bacterial and phage enzymes share two areas oflimited sequence homology with FEN-1, which are termed the N(N-terminal) and I (intermediate) regions, with the residue similaritiesconcentrated around seven conserved acidic residues. Based on crystalstructures of T4 RNase H and T5 exonuclease as well as mutagenesis data,it has been proposed that these residues bind to two Mg²⁺ ions that arerequired for affecting DNA hydrolysis; however, the role each metalplays in the catalytic cycle, which is subtly different for each enzyme,is not well understood (Reviewed in Hosfield et al., 1998b, supra).

[0190] fen-1 genes encoding FEN-1 enzymes useful in the inventioninclude murine fen-1, human fen-1, rat fen-1, Xenopus laevis fen-1, andfen-1 genes derived from four archaebacteria Archaeglobus fulgidus,Methanococcus jannashii, Pyrococcus furiosus and Pyrococcus horikoshii.cDNA clones encoding FEN-1 enzymes have been isolated from human(GenBank Accession Nos.: NM_(—)004111 and L37374), mouse (GenBankAccession No.: L26320), rat (GenBank Accession No.: AA819793), Xenopuslaevis (GenBank Accession Nos.: U68141 and U64563), and P. furiosus(GenBank Accession No.: AF013497). The complete nucleotide sequence forP. horikoshii flap endonuclease has also been determined (GenBankAccession No.: AB005215). The FEN-1 family also includes theSaccharomyces cerevisiae RAD27 gene (GenBank Accession No.: Z28113Y13137) and the Saccharomyces pombe RAD2 gene (GenBank Accession No.:X77041). The archaeal genome of Methanobacterium thermautotrophiculumhas also been sequenced. Although the sequence similarity between FEN-1and prokaryotic and viral 5′→3′ exonucleases is low, FEN-1s within theeukaryotic kingdom are highly conserved at the amino acid level, withthe human and S. cerevisiae proteins being 60% identical and 78%similar. The three archaebacterial FEN-1 proteins are also, highlyhomologous to the eukaryotic FEN-1 enzymes (Reviewed in Matsui et al.,1999., J. Biol. Chem., 274:18297, Hosfield et al., 1998b, J. Biol.Chem., 273:27154 and Lieber, 1997, BioEssays, 19:233).

[0191] The sequence similarities in the two conserved nuclease domains(N-terminal or N and intermediate or I domains) between human and otherFEN-1 family members are 92% (murine), 79% (S. cerevisiae), 77% (S.pombe), 72% (A. fulgidus), 76% (M. jannaschii), and 74% (P. furiosus).

[0192] FEN-1 specifically recognizes the backbone of a 5′single-stranded flap strand and migrates down this flap arm to thecleavage site located at the junction between the two strands of duplexDNA and the single-stranded arm. If the strand upstream of the flap(sometimes called the flap adjacent strand or primer strand) is removed,the resulting structure is termed a pseudo-Y (see FIG. 1). Thisstructure is cleaved by FEN-1, but at 20- to 100-fold lower efficiency.FEN-1 does not cleave 3′ single-stranded flaps. However, FEN-1 acting asan exonuclease will hydrolyze dsDNA substrates containing a gap or nick(Reviewed in Hosfield et al., 1998a, supra, Hosfield et al., 1999b,supra and Lieber 1997, supra). Exonucleolytically, FEN-1 acts at a nickand, with lower efficiency, at a gap or a recessed 5′ end on dsDNA. Atgapped structures, the efficiency of FEN-1 binding and cutting decreaseswith increasing gap size up to approximately five nucleotides and thenstabilizes at a level of cleavage that is equivalent to activity on arecessed 5′ end within dsDNA. Blunt dsDNA, recessed 3′ ends and ssDNAare not cleaved (Reviewed in Lieber 1997, supra). The cleavage activityof FEN enzymes are described in Yoon et al., 1999, Biochemistry, 38:4809; Rao, 1998, J. Bacteriol., 180:5406 and Hosfield et al., 1998,Cell, 95:135-146, incorporated herein by reference.

[0193] FEN nucleases that are useful according to the invention havebeen isolated from a variety of organisms including human (GenBankAccession Nos.: NM_(—)004111 and L37374), mouse (GenBank Accession No.:L26320), rat (GenBank Accession No.: AA819793), yeast (GenBank AccessionNo.: Z28113 Y13137 and GenBank Accession No.: X77041) and xenopus laevis(GenBank Accession Nos.: U68141 and U64563). Such enzymes can be clonedand overexpressed using conventional techniques well known in the art.

[0194] A FEN nuclease according to the invention is preferablythermostable. Thermostable FEN nucleases have been isolated andcharacterized from a variety of thermostable organisms including fourarcheaebacteria. The cDNA sequence (GenBank Accession No.: AF013497) andthe amino acid sequence (Hosfield et al., 1998a, supra and Hosfield etal., 1998b) for P. furiosus flap endonuclease have been determined. Thecomplete nucleotide sequence (GenBank Accession No.: AB005215) and theamino acid sequence (Matsui et al., supra) for P. horikoshii flapendonuclease have also been determined. The amino acid sequence for M.jannaschii (Hosfield et al., 1998b and Matsui et al., 1999 supra) and A.fulgidus (Hosfield et al., 1998b) flap endonuclease have also beendetermined.

[0195] Thermostable FENI enzymes can be cloned and overexpressed usingtechniques well known in the art and described in Hosfield et al.,1998a, supra, Hosfield et al., 1998b, Kaiser et al., 1999, J. Biol.Chem., 274: 21387 and Matusi et al., supra and herein in Example 2entitled “Cloning Pfu FEN-1”.

[0196] The endonuclease activity of a FEN enzyme can be measured by avariety of methods including the following.

[0197] A.FEN Endonuclease Activity Assay

[0198] 1. Templates (for example as shown in FIG. 2) are used toevaluate the activity of a FEN nuclease according to the invention.

[0199] Template 1 is a 5′³³P labeled oligonucleotide (Heltest4 ) withthe following sequence: 5′AAAATAAATAAAAAAAATACTGTTGGGAAGGGCGATCGGTGCG3′. The underlined section of Heltest4 represents the regioncomplementary to M13mp18+. The cleavage product is an 18 nucleotidefragment with the sequence AAAATAAATAAAAAAAAT.

[0200] Heltest4 binds to M13 to produce a complementary double strandeddomain as well as a non-complementary 5′ overhang. This duplex formstemplate 2 (FIG. 2) which is also used for helicase assays. Template 3(FIG. 2) has an additional primer (FENAS) bound to M13 and is directlyadjacent to Heltest 4. The sequence of FENAS is: 5′CCATTCGCCATTCAGGCTGCGCA 3′. In the presence of template 3, FEN binds thefree 5′ terminus of Heltest4, migrates to the junction and cleavesHeltest4 to produce an 18 nucleotide fragment. Templates 1 and 2 serveas controls, although template 2 can also serve as a template.

[0201] Templates are prepared as described below: Template 1 Template 2Template 3 Heltest4 14 μl 14 μl 14 μl M13 ** 14 μl 14 μl FENAS ** ** 14μl H₂O 28 μl 14 μl ** 10x Pfu Buff. 4.6 μl 4.6 μl 4.6 μl

[0202] 10×Pfu buffer is available from Stratagene (Catalog #200536).According to the method of the invention, 10×Pfu buffer is diluted suchthat a reaction is carried out in the presence of 1×buffer.

[0203] M13 is M13mp18+ strand and is at a concentration of 200 ng/μL,³³P labeled Heltest4 is at an approximate concentration of 0.7 ng/μl,and FENAS is at a concentration of 4.3 ng/μl. Based on theseconcentrations, the Heltest4 and M13 are at approximately equal molaramounts (5×10⁻¹⁴) and FENAS is present in an approximately 10×molarexcess (6×10⁻¹³).

[0204] The template mixture is heated at 95° C. for five minutes, cooledto room temperature for 45 minutes and stored at 4° C. overnight.

[0205] 2 μl of FEN-1 or, as a control, H₂O are mixed with the threetemplates as follows:

[0206] 3 μl template

[0207] 0.7 μl 10×cloned Pfu buffer

[0208] 0.56 μl 100 mM MgCl₂

[0209] 2.00 μl enzyme or H₂O

[0210] 0.74 μl H₂O

[0211]7.00 μl total volume

[0212] The reactions are allowed to proceed for 30 minutes at 50° C. andstopped by the addition of 2 μl formamide “Sequencing Stop” solution toeach sample. Samples are heated at 95° C. for five minutes and loaded ona 6% acrylamide, 7M urea CastAway (Stratagene) gel.

[0213] Alternatively, FEN activity can be analyzed in the followingbuffer wherein a one hour incubation time is utilized.

[0214] 10×FEN Buffer

[0215] 500 mM Tris-HCl pH 8.0

[0216] 100 mM MgCl₂

[0217] The reaction mixture below is mixed with 2 μl of FEN or, as acontrol, 2 μl of H₂O.

[0218] 3 μl template

[0219] 0.7 μl 10×FEN buffer

[0220] 2.00 μl enzyme or H₂O

[0221] 1.3 μl H₂O

[0222]7.00 μl total volume

[0223] Samples are incubated for one hour at 50° C. in a Robocyler 96hot top thermal cycler. Following the addition of 2 μl of SequencingStop dye solution, samples are heated at 99° C. for five minutes.Samples are loaded on an eleven-inch long, hand-poured, 20%acrylamide/bis acrylamide, 7M urea gel. The gel is run at 20 watts untilthe bromophenol blue has migrated approximately ⅔ the total distance.The gel is removed from the glass plates and soaked for 10 minutes infix (15% methanol, 5% acetic acid) and then for 10 minutes in water. Thegel is placed on Whatmann 3 mm paper, covered with plastic wrap anddried for 2 hours in a heated vacuum gel dryer. The gel is exposedovernight to X-ray film.

[0224] 2. FEN endonuclease activity can also be measured according tothe method of Kaiser et al., supra). Briefly, reactions are carried outin a 10 μl volume containing 10 mM MOPS, pH 7.5, 0.05% Tween 20, 0.05%Nonidet P-40, 10 μg/ml tRNA, and 200 mM KCl for TaqPol and TthPol or 50mM KCl for all other enzymes. Reaction conditions can be varieddepending on the cleavage structure being analyzed. Substrates (2 μM)and varying amounts of enzyme are mixed with the indicated (above)reaction buffer and overlaid with Chill-out (MJ Research) liquid wax.Substrates are heat denatured at 90° C. for 20 s and cooled to 50° C.,then reactions are started by addition of MgCl₂ or MnCl₂ and incubatedat 50° C. for the specified length of time. Reactions are stopped by theaddition of 10 μl of 95% formamide containing 10 mM EDTA and 0.02%methyl violet (Sigma). Samples are heated to 90° C. for 1 minimmediately before electrophoresis on a 20% denaturing acrylamide gel(19:1 cross-linked), with 7M urea, and in a buffer of 45 mM Tris borate,pH 8.3, 1.4 mM EDTA. Unless otherwise indicated, 1 μl of each stoppedreaction is loaded per lane. Gels are scanned on an FMBIO-100fluorescent gel scanner (Hitachi) using a 505-nm filter. The fraction ofcleaved product is determined from intensities of bands corresponding touncut and cut substrate with FMBIO Analysis software (version 6.0,Hitachi). The fraction of cut product should not exceed 20% to ensurethat measurements approximate initial cleavage rates. The cleavage rateis defined as the concentration of cut product divided by the enzymeconcentration and the time of the reaction (in minutes). For each enzymethree data points are used to determine the rate and experimental error.

[0225] 3. FEN endonuclease activity can also be measured according tothe method of Hosfield et al., 1998a, supra. Briefly, in a final volumeof 13 μl, varying amounts of FEN and 1.54 pmol of labeled cleavagesubstrate are incubated at different temperatures for 30 min before thereaction is quenched with an equal volume of stop solution (10 mM EDTA,95% deionized formamide, and 0.008% bromophenol blue and xylene cyanol).Samples are electrophoresed through denaturing 15% polyacrylamide gels,and the relative amounts of starting material and product arequantitated using the IPLabGel system (Stratagene) running MacBAS imageanalysis software. Most reactions are performed in standard assay buffer(10 mM Tris-HCl (pH 8.0), 10 mM MgCl₂, and 50 μg/ml bovine serumalbumin); however, in a series of experiments the effect of differentdivalent metals and pH levels are studied by varying the standardbuffer. For divalent metals, MgCl₂ is omitted, and different metal ionsare used at a final concentration of 10 mM. To study the influence ofpH, buffers containing different amounts of Tris-HCl, glycine, andsodium acetate are used at a final concentration of 10 mM to obtain awide range of pH levels at 25° C.

[0226] 4. FEN endonuclease activity can also be measured according tothe method of Matusi et al., 1999, supra. Briefly, the enzyme reactionsare performed in a 15-μl reaction mixture containing 50 mM Tris-HCl (pH7.4), 1.5 mM MgCl₂, 0.5 mM β-mercaptoethanol, 100 μg/ml bovine serumalbumin, and 0.6 pmol of a labeled cleavage structure. After incubationfor 30 min at 60° C., the reaction is terminated by adding 15 μl of 95%formamide containing 10 mM EDTA and 1 mg/ml bromphenol blue. The samplesare heated at 95° C. for 10 min, loaded onto a 15% polyacrylamide gel(35 cm×42.5 cm) containing 7M urea and 10×TBE (89 mM Tris-HCl, 89 mMboric acid, 2 mM EDTA (pH 8.0)), and then electrophoresed for 2 h at2000 V. Reaction products are visualized and quantified using aPhosphorImager (Bio-Rad). Size marker, oligonucleotides are 5′end-labeled with [γ-³²P]ATP and T4 polynucleotide kinase.

[0227] To determine the optimum pH, the reaction is performed in anassay mixture (15 μl) containing 1.5 mM MgCl₂, 0.5 mM β-mercaptoethanol,100 μg/ml bovine serum albumin, and 0.6 pmol of 5′ end-labeled cleavagestructure in 50 mM of one of the following buffers at 60° C. for 30 min.Three different 50 mM buffers are used to obtain a wide pH range asfollows: sodium acetate buffer (pH 4.0-5.5), phosphate buffer (pH5.5-8.0), and borate buffer (pH 8.0-9.4).

[0228] B. FEN Exonuclease Activity Assay

[0229] The exonuclease activity of a FEN nuclease according to theinvention can be measured by the method of measuring FEN-1 endonucleaseactivity described in Matsui et al., 1999, supra and summarized above.

[0230] Alternatively, the exonuclease activity of a FEN enzyme can beanalyzed by the method described in Hosfield et al., 1998b, supra.Briefly, exonuclease activities are assayed using a nicked substrate ofFEN under conditions identical to those described for the endonucleaseassays (described above).

[0231] The precise positions of DNA cleavage in both the exonuclease andendonuclease experiments can be obtained by partial digestion of a 5′³²P-labeled template strand using the 3′-5′ exonuclease activity ofKlenow fragment.

[0232] A cleavage structure according to one embodiment of the inventioncomprises a partially displaced 5′ end of an oligonucleotide probeannealed to a target nucleic acid. Another cleavage structure accordingto the invention comprises a target nucleic acid (for example B in FIG.4), an upstream oligonucleotide probe according to the invention, andcomprising a region or regions that are complementary to the targetsequence (for example A in FIG. 4), and a downstream oligonucleotidethat is complementary to the target sequence (for example C in FIG. 4).A cleavage structure according to the invention can be formed by overlapbetween the upstream oligonucleotide and the downstream probe, or byextension of the upstream oligonucleotide by the synthetic activity of anucleic acid polymerase, and subsequent partial displacement of the 5′end of the downstream oligonucleotide. A cleavage structure of this typeis formed according to the method described in the section entitled“Cleavage Structure”.

[0233] Alternatively, a cleavage structure according to the invention isformed by annealing a target nucleic acid to an oligonucleotide probeaccording to the invention wherein the oligonucleotide probe comprises aregion or regions that are complementary to the target nucleic acid, anda non-complementary region that does not anneal to the target nucleicacid and forms a 5′ flap. According to this embodiment, a cleavagestructure comprises a 5′ flap formed by a non-complementary region ofthe oligonucleotide.

[0234] A cleavage structure according to the invention also comprises anoverlapping flap wherein the 3′ end of an upstream oligonucleotidecapable of annealing to a target nucleic acid (for example A in FIG. 4)is complementary to 1 (or more) base pair of the downstreamoligonucleotide probe according to the invention (for example C in FIG.4) that is annealed to a target nucleic acid and wherein the 1 (or more)base pair overlap is directly downstream of the point of extension ofthe single stranded flap and is formed according to method described inthe section entitled “Cleavage Structure”. In one embodiment, theupstream oligonucleotide and the downstream probe hybridize tonon-overlapping regions of the target nucleic acid. In anotherembodiment, the upstream oligonucleotide and the downstream probehybridize to adjacent regions of the target nucleic acid.

[0235] II. Nucleic Acid Polymerases

[0236] The invention provides for nucleic acid polymerases. Preferably,the nucleic acid polymerase according to the invention is thermostable.

[0237] Known DNA polymerases useful according to the invention include,for example, E. coli DNA polymerase I, Thermus thermophilus (Tth) DNApolymerase, Bacillus stearothermophilus DNA polymerase, Thermococcuslitoralis DNA polymerase, Thermus aquaticus (Taq) DNA polymerase andPyrococcus furiosus (Pfu) DNA polymerase.

[0238] Nucleic acid polymerases substantially lacking 5′ to 3′exonuclease activity useful according to the invention include but arenot limited to Klenow and Klenow exo-, and T7 DNA polymerase(Sequenase).

[0239] Thermostable nucleic acid polymerases substantially lacking 5′ to3′ exonuclease activity useful according to the invention include butare not limited to Pfu, exo- Pfu (a mutant form of Pfu that lacks 3′ to5′ exonuclease activity), the Stoffel fragment of Taq, N-truncated Bst,N-truncated Bca, Genta, JdF3 exo-, Vent, Vent exo- (a mutant form ofVent that lacks 3′ to 5′ exonuclease activity), Deep Vent, Deep Ventexo- (a mutant form of Deep Vent that lacks 3′ to 5′ exonucleaseactivity), UlTma, and ThermoSequenase.

[0240] Nucleic acid polymerases useful according to the inventioninclude both native polymerases as well as polymerase mutants, whichlack 5′ to 3′ exonuclease activity. Nucleic acid polymerases usefulaccording to the invention can possess different degrees ofthermostability. Preferably, a nucleic acid polymerase according to theinvention exhibits strand displacement activity at the temperature atwhich it can extend a nucleic acid primer. In a preferred embodiment ofthe invention, a nucleic acid polymerase lacks both 5′ to 3′ and 3′ to5′ exonuclease activity.

[0241] Additional nucleic acid polymerases substantially lacking 5′ to3′ exonuclease activity with different degrees of thermostability usefulaccording to the invention are listed below.

[0242] A. Bacteriophage DNA polymerases (Useful for 37° C. Assays)

[0243] Bacteriophage DNA polymerases are devoid of 5′ to 3′ exonucleaseactivity, as this activity is encoded by a separate polypeptide.Examples of suitable DNA polymerases are T4, T7, and φ29 DNA polymerase.The enzymes available commercially are: T4 (available from many sourcese.g., Epicentre) and T7 (available from many sources, e.g. Epicentre forunmodified and USB for 3′ to 5′ exo⁻T7 “Sequenase” DNA polymerase).

[0244] B. Archaeal DNA polymerases

[0245] There are 2 different classes of DNA polymerases which have beenidentified in archaea: 1. Family B/pol α type (homologs of Pfu fromPyrococcus furiosus) and 2. pol II type (homologs of P. furiosus DP1/DP22-subunit polymerase). DNA polymerases from both classes have been shownto naturally lack an associated 5′ to 3′ exonuclease activity and topossess 3′ to 5′ exonuclease (proofreading) activity. Suitable DNApolymerases (pol α or pol II) can be derived from archaea with optimalgrowth temperatures that are similar to the desired assay temperatures.Examples of suitable archaea include, but are not limited to:

[0246] 1. Thermolabile (useful for 37° C. assays)—e.g., Methanococcusvoltae

[0247] 2. Thermostable (useful for non-PCR assays)—e.g., Sulfolobussolfataricus, Sulfolobus acidocaldarium, Methanococcus jannaschi,Thermoplasma acidophilum. It is estimated that suitable archaea exhibitmaximal growth temperatures of ≦80-85° C. or optimal growth temperaturesof ≦70-80° C.

[0248] 3. Thermostable (useful for PCR assays)—e.g., Pyrococcus species(furiosus, species GB-D, species strain KOD1, woesii, abysii,horikoshii), Thermococcus species (litoralis, species 9°North-7, speciesJDF-3, gorgonarius), Pyrodictium occultum, and Archaeoglobus fulgidus.It is estimated that suitable archaea would exhibit maximal growthtemperatures of ≧80-85° C. or optimal growth temperatures of ≧70-80° C.Appropriate PCR enzymes from the archaeal pol α DNA polymerase group arecommercially available, including KOD (Toyobo), Pfx (Life Technologies,Inc.), Vent (New England BioLabs), Deep Vent (New England BioLabs), andPwo (Boehringer-Mannheim).

[0249] Additional archaea related to those listed above are described inthe following references: Archaea: A Laboratory Manual (Robb, F. T. andPlace, A. R., eds.), Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., 1995 and Thermophilic Bacteria (Kristjansson, J. K.,ed.)CRC Press, Inc., Boca Raton, Fla., 1992.

[0250] C. Eubacterial DNA polymerases

[0251] There are 3 classes of eubacterial DNA polymerases, pol I, II,and III. Enzymes in the Pol I DNA polymerase family possess 5′ to 3′exonuclease activity, and certain members also exhibit 3′ to 5′exonuclease activity. Pol II DNA polymerases naturally lack 5′ to 3′exonuclease activity, but do exhibit 3′ to 5′ exonuclease activity. PolIII DNA polymerases represent the major replicative DNA polymerase ofthe cell and are composed of multiple subunits. The pol III catalyticsubunit lacks 5′ to 3′ exonuclease activity, but in some cases 3′ to 5′exonuclease activity is located in the same polypeptide.

[0252] There are no commercial sources of eubacterial pol II and pol IIIDNA polymerases. There are a variety of commercially available Pol I DNApolymerases, some of which have been modified to reduce or abolish 5′ to3′ exonuclease activity. Methods used to eliminate 5′ to 3′ exonucleaseactivity of pol I DNA polymerases include:

[0253] mutagenesis (as described in Xu et al., 1997, J. Mol. Biol.,268:284 and Kim et al., 1997, Mol. Cells, 7:468).

[0254] N-truncation by proteolytic digestion (as described in Klenow etal., 1971, Eur. J. Biochem., 22: 371), or

[0255] N-truncation by cloning and expressing as C-terminal fragments(as described in Lawyer et al., 1993, PCR Methods Appl., 2:275).

[0256] As for archaeal sources, the assay-temperature requirementsdetermine which eubacteria should be used as a source of a DNApolymerase useful according to the invention (e.g., mesophiles,thermophiles, hyperthermophiles).

[0257] 1. Mesophilic/thermolabile (Useful for 37° C. Assays)

[0258] i. DNA polymerases naturally substantially lacking 5′ to 3′exonuclease activity: pol II or the pol III catalytic subunit frommesophilic eubacteria, such as Escherchia coli, Streptococcuspneumoniae, Haemophilus influenza, Mycobacterium species (tuberculosis,leprae)

[0259] ii. DNA polymerase mutants substantially lacking 5′ to 3′exonuclease activity: Pol I DNA polymerases for N-truncation ormutagenesis can be isolated from the mesophilic eubacteria listed above(Ci). A commercially-available eubacterial DNA polymerase pol I fragmentis the Klenow fragment (N-truncated E. coli pol I; Stratagene).

[0260] 2. Thermostable (Useful for non PCR assays)

[0261] i. DNA polymerases naturally substantially lacking 5′ to 3′exonuclease activity: Pol II or the pol III catalytic subunit fromthermophilic eubacteria, such as Bacillus species (e.g.,stearothermophilus, caldotenax, caldovelox)

[0262] ii. DNA polymerase mutants substantially lacking 5′ to 3′exonuclease activity: Suitable pol I DNA polymerases for N-truncation ormutagenesis can be isolated from thermophilic eubacteria such as theBacillus species listed above. Thermostable N-truncated fragments of B.stearothermophilus DNA polymerase pol I are commercially available andsold under the trade names Bst DNA polymerase I large fragment (Bio-Radand Isotherm DNA polymerase (Epicentre)). A C-terminal fragment ofBacillus caldotenax pol I is available from Panvera (sold under thetradename Ladderman).

[0263] 3. Thermostable (Useful for PCR assays)

[0264] i. DNA polymerases naturally substantially lacking 5′ to 3′exonuclease activity: Pol II or pol III catalytic subunit from Thermusspecies (aquaticus, thermophilus, favus, ruber, caldophilus, filiformis,brokianus) or from Thermotoga maritima. The catalytic pol III subunitsfrom Thermus thermophilus and Thermus aquaticus are described in Yi-Pinget al., 1999, J. Mol. Evol., 48:756 and McHenry et al., 1997, J. Mol.Biol., 272:178.

[0265] ii. DNA polymerase mutants substantially lacking 5′ to 3′exonuclease activity: Suitable pol I DNA polymerases for N-truncation ormutagenesis can be isolated from a variety of thermophilic eubacteria,including Thermus species and Thermotoga maritima (see above).Thermostable fragments of Thermus aquaticus DNA polymerase pol I (Taq)are commercially available and sold under the trade names KlenTaq1 (AbPeptides), Stoffel fragment (Perkin-Elmer), and ThermoSequenase(Amersham). In addition to C-terminal fragments, 5′ to 3′ exonuclease⁻Taq mutants are also commercially available, such as TaqFS(Hoffman-LaRoche). In addition to 5′-3′ exonuclease⁻ versions of Taq, anN-truncated version of Thermotoga maritima DNA polymerase I is alsocommercially available (tradename UlTma, Perkin-Elmer).

[0266] Additional eubacteria related to those listed above are describedin Thermophilic Bacteria (Kristjansson, J. K.,ed.) CRC Press, Inc., BocaRaton, Fla., 1992.

[0267] D. Eukaryotic 5′ to 3′ Exonuclease⁻ DNA polymerases (Useful for37° C. Assays)

[0268] There are several DNA polymerases that have been identified ineukaryotes, including DNA pol α, (replication/repair), δ (replication),ε (replication), β (repair) and γ (mitochondrial replication).Eukaryotic DNA polymerases are devoid of 5′ to 3′ exonuclease activity,as this activity is encoded by a separate polypeptide (e.g., mammalianFEN-1 or yeast RAD2). Suitable thermolabile DNA polymerases may beisolated from a variety of eukaryotes (including but not limited toyeast, mammalian cells, insect cells, Drosophila) and eukaryotic viruses(e.g., EBV, adenovirus).

[0269] It is possible that DNA polymerase mutants lacking 3′-5′exonuclease (proofreading) activity, in addition to lacking 5′ to 3′exonuclease activity, could exhibit improved performance in FEN-baseddetection strategies. For example, reducing or abolishing inherent 3′ to5′ exonuclease activity may lower background signals by diminishingnon-specific exonucleolytic degradation of labeled probes. Three 3′ to5′ exonuclease motifs have been identified, and mutations in theseregions have been shown to abolish 3′ to 5′ exonuclease activity inKlenow, φ29, T4, T7, and Vent DNA polymerases, yeast Pol α, Pol β, andPol γ, and Bacillus subtilis Pol III (reviewed in Derbeyshire et al.,1995, Methods. Enzymol. 262:363). Methods for preparing additional DNApolymerase mutants, with reduced or abolished 3′ to 5′ exonucleaseactivity, are well known in the art.

[0270] Commercially-available enzymes that lack both 5′ to 3′ and 3′ to5′ exonuclease activities include Sequenase (exo⁻ T7; USB), Pfu exo⁻(Stratagene), exo⁻ Vent (New England BioLabs), exo⁻ DeepVent (NewEngland BioLabs), exo⁻ Klenow fragment (Stratagene), Bst (Bio-Rad),Isotherm (Epicentre), Ladderman (Panvera), KlenTaq1 (Ab Peptides),Stoffel fragment (Perkin-Elmer), ThermoSequenase (USB), and TaqFS(Hoffman-LaRoche).

[0271] If polymerases other than Pfu are used, buffers and extensiontemperatures are selected to allow for optimal activity by theparticular polymerase useful according to the invention. Buffers andextension temperatures useful for polymerases according to the inventionare know in the art and can also be determined from the Vendor'sspecifications.

[0272] III. Nucleic Acids

[0273] A. Nucleic Acid Sequences Useful in the Invention

[0274] The invention provides for methods of detecting or measuring atarget nucleic acid; and also utilizes oligonucleotides, primers andprobes for forming a cleavage structure according to the invention andprimers for amplifying a template nucleic acid sequence.

[0275] As used herein, the terms “nucleic acid”, “polynucleotide” and“oligonucleotide” refer to primers, probes, and oligomer fragments to bedetected, and shall be generic to polydeoxyribonucleotides (containing2-deoxy-D-ribose), to polyribonucleotides (containing D-ribose), and toany other type of polynucleotide which is an N-glycoside of a purine orpyrimidine base, or modified purine or pyrimidine bases (includingabasic sites). There is no intended distinction in length between theterm “nucleic acid”, “polynucleotide” and “oligonucleotide”, and theseterms will be used interchangeably. These terms refer only to theprimary structure of the molecule. Thus, these terms include double- andsingle-stranded DNA, as well as double- and single-stranded RNA.

[0276] The complement of a nucleic acid sequence as used herein refersto an oligonucleotide which, when aligned with the nucleic acid sequencesuch that the 5′ end of one sequence is paired with the 3′ end of theother, is in “antiparallel association.”

[0277] The oligonucleotide is not necessarily physically derived fromany existing or natural sequence but may be generated in any manner,including chemical synthesis, DNA replication, reverse transcription ora combination thereof. The terms “oligonucleotide” or “nucleic acid”intend a polynucleotide of genomic DNA or RNA, cDNA, semisynthetic, orsynthetic origin which, by virtue of its synthetic origin ormanipulation: (1) is not associated with all or a portion of thepolynucleotide with which it is associated in nature; and/or (2) islinked to a polynucleotide other than that to which it is linked innature.

[0278] Because mononucleotides are reacted to make oligonucleotides in amanner such that the 5′ phosphate of one mononucleotide pentose ring isattached to the 3′ oxygen of its neighbor in one direction via aphosphodiester linkage, an end of oligonucleotide is referred to as the“5′ end” if its 5′ phosphate is not linked to the 3′ oxygen of amononucleotide pentose ring and as the “3′ end” if its 3′ oxygen is notlinked to a 5′ phosphate of a subsequent mononucleotide pentose ring. Asused herein, a nucleic acid sequence, even if internal to a largeroligonucleotide, also may be said to have 5′ and 3′ ends.

[0279] When two different, non-overlapping oligonucleotides anneal todifferent regions of the same linear complementary nucleic acidsequence, and the 3′ end of one oligonucleotide points toward the 5′ endof the other, the former may be called the “upstream” oligonucleotideand the latter the “downstream” oligonucleotide.

[0280] Certain bases not commonly found in natural nucleic acids may beincluded in the nucleic acids of the present invention and include, forexample, inosine and 7-deazaguanine. Complementarity need not beperfect; stable duplexes may contain mismatched base pairs or unmatchedbases. Those skilled in the art of nucleic acid technology can determineduplex stability empirically considering a number of variablesincluding, for example, the length of the oligonucleotide, basecomposition and sequence of the oligonucleotide, ionic strength, andincidence of mismatched base pairs.

[0281] Stability of a nucleic acid duplex is measured by the meltingtemperature, or “T_(m)”. The T_(m) of a particular nucleic acid duplexunder specified conditions is the temperature at which half of the basepairs have disassociated.

[0282] B. Primers and Probes Useful According to the Invention

[0283] The invention provides for oligonucleotide primers and probesuseful for detecting or measuring a nucleic acid, for amplifying atemplate nucleic acid sequence, and for forming a cleavage structureaccording to the invention.

[0284] The term “primer” may refer to more than one primer and refers toan oligonucleotide, whether occurring naturally, as in a purifiedrestriction digest, or produced synthetically, which is capable ofacting as a point of initiation of synthesis along a complementarystrand when placed under conditions in which synthesis of a primerextension product which is complementary to a nucleic acid strand iscatalyzed. Such conditions include the presence of four differentdeoxyribonucleoside triphosphates and a polymerization-inducing agentsuch as DNA polymerase or reverse transcriptase, in a suitable buffer(“buffer” includes substituents which are cofactors, or which affect pH,ionic strength, etc.), and at a suitable temperature. The primer ispreferably single-stranded for maximum efficiency in amplification.

[0285] Oligonucleotide primers useful according to the invention aresingle-stranded DNA or RNA molecules that are hybridizable to a templatenucleic acid sequence and prime enzymatic synthesis of a second nucleicacid strand. The primer is complementary to a portion of a targetmolecule present in a pool of nucleic acid molecules. It is contemplatedthat oligonucleotide primers according to the invention are prepared bysynthetic methods, either chemical or enzymatic. Alternatively, such amolecule or a fragment thereof is naturally-occurring, and is isolatedfrom its natural source or purchased from a commercial supplier.Oligonucleotide primers are 5 to 100 nucleotides in length, ideally from17 to 40 nucleotides, although primers of different length are of use.Primers for amplification are preferably about 17-25 nucleotides.Primers for the production of a cleavage structure according to theinvention are preferably about 17-45 nucleotides. Primers usefulaccording to the invention are also designed to have a particularmelting temperature (Tm) by the method of melting temperatureestimation. Commercial programs, including Oligo™, Primer Design andprograms available on the internet, including Primer3 and OligoCalculator can be used to calculate a Tm of a nucleic acid sequenceuseful according to the invention. Preferably, the Tm of anamplification primer useful according to the invention, as calculatedfor example by Oligo Calculator, is preferably between about 45 and 65°C. and more preferably between about 50 and 60° C. Preferably, the Tm ofa probe useful according to the invention is 7° C. higher than the Tm ofthe corresponding amplification primers.

[0286] Primers and probes according to the invention can be labeled andcan be used to prepare a labeled cleavage structure. Pairs ofsingle-stranded DNA primers, a DNA primer and a probe or a probe can beannealed to sequences within a target nucleic acid. In certainembodiments, a primer can be used to prime amplifying DNA synthesis of atarget nucleic acid.

[0287] Typically, selective hybridization occurs when two nucleic acidsequences are substantially complementary (at least about 65%complementary over a stretch of at least 14 to 25 nucleotides,preferably at least about 75%, more preferably at least about 90%complementary). See Kanehisa, M., 1984, Nucleic Acids Res. 12: 203,incorporated herein by reference. As a result, it is expected that acertain degree of mismatch at the priming site is tolerated. Suchmismatch may be small, such as a mono-, di- or tri-nucleotide.Alternatively, a region of mismatch may encompass loops, which aredefined as regions in which there exists a mismatch in an uninterruptedseries of four or more nucleotides.

[0288] Numerous factors influence the efficiency and selectivity ofhybridization of the primer to a second nucleic acid molecule. Thesefactors, which include primer length, nucleotide sequence and/orcomposition, hybridization temperature, buffer composition and potentialfor steric hindrance in the region to which the primer is required tohybridize, will be considered when designing oligonucleotide primersaccording to the invention.

[0289] A positive correlation exists between primer length and both theefficiency and accuracy with which a primer will anneal to a targetsequence. In particular, longer sequences have a higher meltingtemperature (T_(M)) than do shorter ones, and are less likely to berepeated within a given target sequence, thereby minimizing promiscuoushybridization. Primer sequences with a high G-C content or that comprisepalindromic sequences tend to self-hybridize, as do their intendedtarget sites, since unimolecular, rather than bimolecular, hybridizationkinetics are generally favored in solution. However, it is alsoimportant to design a primer that contains sufficient numbers of G-Cnucleotide pairings since each G-C pair is bound by three hydrogenbonds, rather than the two that are found when A and T bases pair tobind the target sequence, and therefore forms a tighter, stronger bond.Hybridization temperature varies inversely with primer annealingefficiency, as does the concentration of organic solvents, e.g.formamide, that might be included in a priming reaction or hybridizationmixture, while increases in salt concentration facilitate binding. Understringent annealing conditions, longer hybridization probes, orsynthesis primers, hybridize more efficiently than do shorter ones,which are sufficient under more permissive conditions. Preferably,stringent hybridization is performed in a suitable buffer (for example,1×Sentinel Molecular Beacon PCR core buffer, Stratagene Catalog #600500;1×Pfu buffer, Stratagene Catalog #200536; or 1×cloned Pfu buffer,Stratagene Catalog #200532) under conditions that allow the nucleic acidsequence to hybridize to the oligonucleotide primers and/or probes(e.g., 95° C.). Stringent hybridization conditions can vary (for examplefrom salt concentrations of less than about IM, more usually less thanabout 500 mM and preferably less than about 200 mM) and hybridizationtemperatures can range (for example, from as low as 0° C. to greaterthan 22° C., greater than about 30° C., and (most often) in excess ofabout 37° C.) depending upon the lengths and/or the nucleic acidcomposition or the oligonucleotide primers and/or probes. Longerfragments may require higher hybridization temperatures for specifichybridization. As several factors affect the stringency ofhybridization, the combination of parameters is more important than theabsolute measure of a single factor.

[0290] Oligonucleotide primers can be designed with these considerationsin mind and synthesized according to the following methods.

[0291] 1. Oligonucleotide Primer Design Strategy

[0292] The design of a particular oligonucleotide primer for the purposeof sequencing or PCR, involves selecting a sequence that is capable ofrecognizing the target sequence, but has a minimal predicted secondarystructure. The oligonucleotide sequence may or may not bind only to asingle site in the target nucleic acid. Furthermore, the Tm of theoligonucleotide is optimized by analysis of the length and GC content ofthe oligonucleotide. Furthermore, when designing a PCR primer useful forthe amplification of genomic DNA, the selected primer sequence does notdemonstrate significant matches to sequences in the GenBank database (orother available databases).

[0293] The design of a primer useful according to the invention, isfacilitated by the use of readily available computer programs, developedto assist in the evaluation of the several parameters described aboveand the optimization of primer sequences. Examples of such programs are“PrimerSelect” of the DNAStar™ software package (DNAStar, Inc.; Madison,Wis.), OLIGO 4.0 (National Biosciences, Inc.), PRIMER, OligonucleotideSelection Program, PGEN and Amplify (described in Ausubel et al., 1995,Short Protocols in Molecular Biology, 3rd Edition, John Wiley & Sons).In one embodiment, primers are designed with sequences that serve astargets for other primers to produce a PCR product that has knownsequences on the ends which serve as targets for further amplification(e.g. to sequence the PCR product). If many different target nucleicacids are amplified with specific primers that share a common ‘tail'sequence’, the PCR products from these distinct genes can subsequentlybe sequenced with a single set of primers. Alternatively, in order tofacilitate subsequent cloning of amplified sequences, primers aredesigned with restriction enzyme site sequences appended to their 5′ends. Thus, all nucleotides of the primers are derived from a targetnucleic acid or sequences adjacent to a target nucleic acid, except forthe few nucleotides necessary to form a restriction enzyme site. Suchenzymes and sites are well known in the art. If the genomic sequence ofa target nucleic acid and the sequence of the open reading frame of atarget nucleic acid are known, design of particular primers is wellwithin the skill of the art.

[0294] It is well known by those with skill in the art thatoligonucleotides can be synthesized with certain chemical and/or capturemoieties, (including capture elements as defined herein) such that theycan be coupled to solid supports and bind to a binding moiety or tag, asdefined herein. Suitable capture elements include, but are not limitedto a nucleic acid binding protein or a nucleotide sequence. Suitablecapture elements include, but are not limited to, biotin, a hapten, aprotein, or a chemically reactive moiety. Such oligonucleotides mayeither be used first in solution, and then captured onto a solidsupport, or first attached to a solid support and then used in adetection reaction. An example of the latter would be to couple adownstream probe molecule to a solid support, such that the 5′ end ofthe downstream probe molecule comprised a fluorescent quencher. The samedownstream probe molecule would also comprise a fluorophore in alocation such that a FEN nuclease cleavage would physically separate thequencher from the fluorophore. For example, the target nucleic acidcould hybridize with the solid-phase downstream probe oligonucleotide,and a liquid phase upstream primer could also hybridize with the targetmolecule, such that a FEN cleavage reaction occurs on the solid supportand liberates the 5′ quencher moiety from the complex. This would causethe solid support-bound fluorophore to be detectable, and thus revealthe presence of a cleavage event upon a suitably labeled or identifiedsolid support. Different downstream probe molecules could be bound todifferent locations on an array. The location on the array wouldidentify the probe molecule, and indicate the presence of the templateto which the probe molecule can hybridize.

[0295] 2. Synthesis

[0296] The primers themselves are synthesized using techniques that arealso well known in the art. Methods for preparing oligonucleotides ofspecific sequence are known in the art, and include, for example,cloning and restriction digest analysis of appropriate sequences anddirect chemical synthesis. Once designed, oligonucleotides are preparedby a suitable chemical synthesis method, including, for example, thephosphotriester method described by Narang et al., 1979, Methods inEnzymology, 68:90, the phosphodiester method disclosed by Brown et al.,1979, Methods in Enzymology, 68:109, the diethylphosphoramidate methoddisclosed in Beaucage et al., 1981, Tetrahedron Letters, 22:1859, andthe solid support method disclosed in U.S. Pat. No. 4,458,066, or byother chemical methods using either a commercial automatedoligonucleotide synthesizer (which is commercially available) or VLSIPSTtechnology.

[0297] C. Probes

[0298] The invention provides for probes useful for forming a cleavagestructure or a labeled cleavage structure as defined herein. Methods ofpreparing a labeled cleavage structure according to the invention areprovided in the section entitled “Cleavage Structure” below.

[0299] As used herein, the term “probe” refers to a probe having asecondary structure that changes upon binding of the probe to a targetnucleic acid and comprising a binding moiety, as defined herein, whereinthe probe forms a duplex structure with a sequence in the target nucleicacid, due to complementarity of at least one sequence in the probe witha sequence in the target region. A probe according to the invention canalso be labeled. The probe, preferably, does not contain a sequencecomplementary to sequence(s) used in the primer extension(s). Generallythe 3′ terminus of the probe will be “blocked” to prohibit incorporationof the probe into a primer extension product. Methods of labeling aprobe according to the invention and suitable labels are described belowin the section entitled “Cleavage Structure”.

[0300] The general design of a probe according to the invention isdescribed in the section entitled, “Primers and Probes Useful Accordingto the Invention”. Typically, a probe according to the inventioncomprises a target nucleic acid binding sequence that is from about7-140 nucleotides, and preferably from about 10-140 nucleotides long (C,FIG. 4). A probe according to the invention also comprises twocomplementary nucleic acid sequence regions, as defined herein (b andb′, FIG. 4) that are complementary and bind to each other to form aregion of secondary structure in the absence of a target nucleic acid.Regions b and b′ are 3-25 nucleotides, preferably 4-15 nucleotides andmore preferably 5-11 nucleotides in length. The actual length will bechosen with reference to the target nucleic acid binding sequence suchthat the secondary structure of the probe is preferably stable when theprobe is not bound to the target nucleic acid at the temperature atwhich cleavage of a cleavage structure comprising the probe bound to atarget nucleic acid is performed.

[0301] A probe according to the invention is capable of forming asecondary structure as defined herein, (including a stem loop, ahairpin, an internal loop, a bulge loop, a branched structure and apseudoknot) or multiple secondary structures, cloverleaf type structuresor any three-dimensional structure as defined hereinabove.

[0302] For example, according to one embodiment of the presentinvention, a probe can be an oligonucleotide with secondary structuresuch as a hairpin or a stem-loop, and includes, but is not limited tomolecular beacons, safety pins, scorpions, and sunrise/amplifluorprobes.

[0303] Molecular beacon probes comprise a hairpin, or stem-loopstructure which possesses a pair of interactive signal generatinglabeled moieties (e.g., a fluorophore and a quencher) effectivelypositioned to quench the generation of a detectable signal when thebeacon probe is not hybridized to the target nucleic acid. The loopcomprises a region that is complementary to a target nucleic acid. Theloop is flanked by 5′ and 3′ regions (“arms”) that reversibly interactwith one another by means of complementary nucleic acid sequences whenthe region of the probe that is complementary to a nucleic acid targetsequence is not bound to the target nucleic acid. Alternatively, theloop is flanked by 5′ and 3′ regions (“arms”) that reversibly interactwith one another by means of attached members of an affinity pair toform a secondary structure when the region of the probe that iscomplementary to a nucleic acid target sequence is not bound to thetarget nucleic acid. As used herein, “arms” refers to regions of amolecular beacon probe that a) reversibly interact with one another bymeans of complementary nucleic acid sequences when the region of theprobe that is complementary to a nucleic acid target sequence is notbound to the target nucleic acid or b) regions of a probe thatreversibly interact with one another by means of attached members of anaffinity pair to form a secondary structure when the region of the probethat is complementary to a nucleic acid target sequence is not bound tothe target nucleic acid. When a molecular beacon probe is not hybridizedto target, the arms hybridize with one another to form a stem hybrid,which is sometimes referred to as the “stem duplex”. This is the closedconformation. When a molecular beacon probe hybridizes to its target the“arms” of the probe are separated. This is the open conformation. In theopen conformation an arm may also hybridize to the target. Such probesmay be free in solution, or they may be tethered to a solid surface.When the arms are hybridized (e.g., form a stem) the quencher is veryclose to the fluorophore and effectively quenches or suppresses itsfluorescence, rendering the probe dark. Such probes are described inU.S. Pat. Nos. 5,925,517 and 6,037,130.

[0304] As used herein, a molecular beacon probe can also be an“allele-discriminating” probe as described herein.

[0305] Molecular beacon probes have a fluorophore attached to one armand a quencher attached to the other arm. The fluorophore and quencher,for example, tetramethylrhodamine and DABCYL, need not be a FRET pair.

[0306] For stem loop probes useful in this invention, the length of theprobe sequence that is complementary to the target, the length of theregions of a probe (e.g., stem hybrid) that reversibly interact with oneanother by means of complementary nucleic acid sequences, when theregion complementary to a nucleic acid target sequence is not bound tothe target nucleic acid, and the relation of the two, is designedaccording to the assay conditions for which the probe is to be utilized.The lengths of the target-complementary sequences and the stem hybridsequences for particular assay conditions can be estimated according towhat is known in the art. The regions of a probe that reversiblyinteract with one another by means of complementary nucleic acidsequences when the region of the probe that is complementary to anucleic acid target sequence is not bound to the target nucleic acid arein the range of 6 to 100, preferably 8 to 50 nucleotides and mostpreferably 8 to 25 nucleotides each. The length of the probe sequencethat is complementary to the target is preferably 17-40 nucleotides,more preferably 17-30 nucleotides and most preferably 17-25 nucleotideslong.

[0307] The oligonucleotide sequences of molecular beacon probes modifiedaccording to this invention may be DNA, RNA, cDNA or combinationsthereof. Modified nucleotides may be included, for examplenitropyrole-based nucleotides or 2′-O-methylribonucleotides. Modifiedlinkages also may be included, for example phosphorothioates. Modifiednucleotides and modified linkages may also be incorporated inwavelength-shifting primers according to this invention.

[0308] A safety pin probe, as utilized in the present invention,requires a “universal” hairpin probe 1 (FIG. 9, b 171), comprising ahairpin structure, with a fluorophore (FAM) on the 5′ arm of the hairpinand a quencher (Dabeyl) on the 3′ arm, and a probe 2 (FIG. 9, SP170a)comprising a stem-loop comprising two domains: the 5′ two thirds ofprobe 2 have a (universal) sequence complementary to the hairpin probe1, and nucleotides that will stop the DNA polymerase, and the 3′ onethird of probe 2, which serves as the target specific primer. As thepolymerase, primed from the reverse primer (that is, the 3′ one third ofprobe 2) synthesizes the top strand, the 5′ end of probe 2 will bedisplaced and degraded by the 5′ exonucleolytic activity until the “stopnucleotides” are reached. At this time the remainder of probe 2 opens upor unfolds and serves as a target for hairpin probe 1, therebyseparating the fluorophore from the quencher (FIG. 9).

[0309] Scorpion probes, as used in the present invention comprise a 3′primer with a 5′ extended probe tail comprising a hairpin structurewhich possesses a fluorophore/quencher pair. The probe tail is“protected” from replication in the 5′→3′ direction by the inclusion ofhexethlyene glycol (HEG) which blocks the polymerase from replicatingthe probe. During the first round of amplification the 3′target-specific primer anneals to the target and is extended such thatthe scorpion is now incorporated into the newly synthesized strand,which possesses a newly synthesized target region for the 5′ probe.During the next round of denaturation and annealing, the probe region ofthe scorpion hairpin loop will hybridize to the target, thus separatingthe fluorophore and quencher and creating a measurable signal. Suchprobes are described in Whitcombe et al., Nature Biotechnology 17:804-807 (1999), and in FIG. 10.

[0310] An additional oligonucleotide probe useful in the presentinvention is the sunrise/amplifluor probe. The sunrise/amplifluor probeis of similar construction as the scorpion probe with the exception thatis lacks the HEG monomer to block the 5′→3′ replication of the hairpinprobe region. Thus, in the first round of amplification, the 3′ targetspecific primer of the sunrise/amplifluor anneals to the target and isextended, thus incorporating the hairpin probe into the newlysynthesized strand (sunrise strand). During the second round ofamplification a second, non-labeled primer anneals to the 3′ end of thesunrise strand (Cycle 2 in FIG. 11). However, as the polymerase reachesthe 5′ end of the hairpin, due to the lack of the HEG stop sequence, thepolymerase will displace and replicate the hairpin, thus separating thefluorophore and quencher, and incorporating the linearized hairpin probeinto the new strand. Probes of this type are described further inNazarneko et al., Nucleic Acid Res. 25: 2516-2521 (1997), and in FIG.11.

[0311] For safety pin, scorpion and sunrise/amplifluor probes useful inthis invention, the length of the probe sequence that is complementaryto the target, the length of the regions of a probe (e.g., stem hybrid)that reversibly interact with one another by means of complementarynucleic acid sequences when the region complementary to a nucleic acidtarget sequence is not bound to the target nucleic acid and the relationof the two is designed according to the assay conditions for which theprobe is to be utilized. The lengths of the target-complementarysequences and the stem hybrid sequences for particular assay conditionscan be estimated according to what is known in the art. The regions of aprobe that reversibly interact with one another by means ofcomplementary nucleic acid sequences when the region complementary to anucleic acid target sequence is not bound to the target nucleic acid arein the range of 6 to 100, preferably 8 to 50 nucleotides and mostpreferably 8 to 25 nucleotides each. The length of the probe sequencethat is complementary to the target is preferably 17-40 nucleotides,more preferably 17-30 nucleotides and most preferably 17-25 nucleotideslong. The stability of the interaction between the regions of a probethat reversibly interact with one another by means of complementarynucleic acid sequences is determined by routine experimentation toachieve proper functioning. In addition to length, the stability of theinteraction between the regions of a probe that reversibly interact withone another by means of complementary nucleic acid sequences can beadjusted by altering the G-C content and inserting destabilizingmismatches. One of the regions of a probe that reversibly interact withone another by means of complementary nucleic acid sequences can bedesigned to be partially or completely complementary to the target. Ifthe 3′ arm is complementary to the target the probe can serve as aprimer for a DNA polymerase. Also, wavelength-shifting molecular beaconprobes can be immobilized to solid surfaces, as by tethering, or befree-floating.

[0312] A wide range of fluorophores may be used in probes and primersaccording to this invention. Available fluorophores include coumarin,fluorescein, tetrachlorofluorescein, hexachlorofluorescein, Luciferyellow, rhodamine, BODIPY, tetramethylrhodamine, Cy3, Cy5, Cy7, eosine,Texas red and ROX. Combination fluorophores such asfluorescein-rhodamine dimers, described, for example, by Lee et al.(1997), Nucleic Acids Research 25:2816, are also suitable. Fluorophoresmay be chosen to absorb and emit in the visible spectrum or outside thevisible spectrum, such as in the ultraviolet or infrared ranges.

[0313] Suitable quenchers described in the art include particularlyDABCYL and variants thereof, such as DABSYL, DABMI and Methyl Red.Fluorophores can also be used as quenchers, because they tend to quenchfluorescence when touching certain other fluorophores. Preferredquenchers are either chromophores such as DABCYL or malachite green, orfluorophores that do not fluoresce in the detection range when the probeis in the open conformation.

[0314] D. Production of a Nucleic Acid

[0315] The invention provides nucleic acids to be detected and ormeasured, for amplification of a target nucleic acid and for formationof a cleavage structure.

[0316] The present invention utilizes nucleic acids comprising RNA,cDNA, genomic DNA, synthetic forms, and mixed polymers. The inventionincludes both sense and antisense strands of a nucleic acid. Accordingto the invention, the nucleic acid may be chemically or biochemicallymodified or may contain non-natural or derivatized nucleotide bases.Such modifications include, for example, labels, methylation,substitution of one or more of the naturally occurring nucleotides withan analog, internucleotide modifications such as uncharged linkages(e.g. methyl phosphonates, phosphorodithioates, etc.), pendent moieties(e.g., polypeptides), intercalators, (e.g. acridine, psoralen, etc.)chelators, alkylators, and modified linkages (e.g. alpha anomericnucleic acids, etc.) Also included are synthetic molecules that mimicpolynucleotides in their ability to bind to a designated sequence viahydrogen bonding and other chemical interactions. Such molecules areknown in the art and include, for example, those in which peptidelinkages substitute for phosphate linkages in the backbone of themolecule.

[0317] 1. Nucleic Acids Comprising DNA

[0318] a. Cloning

[0319] Nucleic acids comprising DNA can be isolated from cDNA or genomiclibraries by cloning methods well known to those skilled in the art(Ausubel et al., supra). Briefly, isolation of a DNA clone comprising aparticular nucleic acid sequence involves screening a recombinant DNA orcDNA library and identifying the clone containing the desired sequence.Cloning will involve the following steps. The clones of a particularlibrary are spread onto plates, transferred to an appropriate substratefor screening, denatured, and probed for the presence of a particularnucleic acid. A description of hybridization conditions, and methods forproducing labeled probes is included below.

[0320] The desired clone is preferably identified by hybridization to anucleic acid probe or by expression of a protein that can be detected byan antibody. Alternatively, the desired clone is identified bypolymerase chain amplification of a sequence defined by a particular setof primers according to the methods described below.

[0321] The selection of an appropriate library involves identifyingtissues or cell lines that are an abundant source of the desiredsequence. Furthermore, if a nucleic acid of interest contains regulatorysequence or intronic sequence a genomic library is screened (Ausubel etal., supra).

[0322] b. Genomic DNA

[0323] Nucleic acid sequences of the invention are amplified fromgenomic DNA. Genomic DNA is isolated from tissues or cells according tothe following method.

[0324] To facilitate detection of a variant form of a gene from aparticular tissue, the tissue is isolated free from surrounding normaltissues. To isolate genomic DNA from mammalian tissue, the tissue isminced and frozen in liquid nitrogen. Frozen tissue is ground into afine powder with a prechilled mortar and pestle, and suspended indigestion buffer (100 mM NaCl, 10 mM Tris-HCl, pH 8.0, 25 mM EDTA, pH8.0, 0.5% (w/v) SDS, 0.1 mg/ml proteinase K) at 1.2 ml digestion bufferper 100 mg of tissue. To isolate genomic DNA from mammalian tissueculture cells, cells are pelleted by centrifugation for 5 min at 500×g,resuspended in 1-10 ml ice-cold PBS, repelleted for 5 min at 500×g andresuspended in 1 volume of digestion buffer.

[0325] Samples in digestion buffer are incubated (with shaking) for12-18 hours at 50° C., and then extracted with an equal volume ofphenol/chloroform/isoamyl alcohol. If the phases are not resolvedfollowing a centrifugation step (10 min at 1700×g), another volume ofdigestion buffer (without proteinase K) is added and the centrifugationstep is repeated. If a thick white material is evident at the interfaceof the two phases, the organic extraction step is repeated. Followingextraction the upper, aqueous layer is transferred to a new tube towhich will be added 1/2 volume of 7.5M ammonium acetate and 2 volumes of100% ethanol. The nucleic acid is pelleted by centrifugation for 2 minat 1700×g, washed with 70% ethanol, air dried and resuspended in TEbuffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA, pH 8.0) at 1 mg/ml. ResidualRNA is removed by incubating the sample for 1 hour at 37° C. in thepresence of 0.1% SDS and 1 μg/ml DNase-free RNase, and repeating theextraction and ethanol precipitation steps. The yield of genomic DNA,according to this method is expected to be approximately 2 mg DNA/1 gcells or tissue (Ausubel et al., supra). Genomic DNA isolated accordingto this method can be used for PCR analysis, according to the invention.

[0326] c. Restriction Digest (of cDNA or Genomic DNA)

[0327] Following the identification of a desired cDNA or genomic clonecontaining a particular target nucleic acid, nucleic acids of theinvention may be isolated from these clones by digestion withrestriction enzymes.

[0328] The technique of restriction enzyme digestion is well known tothose skilled in the art (Ausubel et al., supra). Reagents useful forrestriction enzyme digestion are readily available from commercialvendors including Stratagene, as well as other sources.

[0329] d. PCR

[0330] Nucleic acids of the invention may be amplified from genomic DNAor other natural sources by the polymerase chain reaction (PCR). PCRmethods are well-known to those skilled in the art.

[0331] PCR provides a method for rapidly amplifying a particular DNAsequence by using multiple cycles of DNA replication catalyzed by athermostable, DNA-dependent DNA polymerase to amplify the targetsequence of interest. PCR requires the presence of a target nucleic acidto be amplified, two single stranded oligonucleotide primers flankingthe sequence to be amplified, a DNA polymerase, deoxyribonucleosidetriphosphates, a buffer and salts.

[0332] PCR, is performed as described in Mullis and Faloona, 1987,Methods Enzymol., 155: 335, herein incorporated by reference.

[0333] The polymerase chain reaction (PCR) technique, is disclosed inU.S. Pat. Nos. 4,683,202, 4,683,195 and 4,800,159. In its simplest form,PCR is an in vitro method for the enzymatic synthesis of specific DNAsequences, using two oligonucleotide primers that hybridize to oppositestrands and flank the region of interest in the target DNA. A repetitiveseries of reaction steps involving template denaturation, primerannealing and the extension of the annealed primers by DNA polymeraseresults in the exponential accumulation of a specific fragment whosetermini are defined by the 5′ ends of the primers. PCR is reported to becapable of producing a selective enrichment of a specific DNA sequenceby a factor of 10⁹. The PCR method is also described in Saiki et al.,1985, Science 230:1350.

[0334] PCR is performed using template DNA (at least 1 fg; moreusefully, 1-1000 ng) and at least 25 pmol of oligonucleotide primers. Atypical reaction mixture includes: 2 μl of DNA, 25 pmol ofoligonucleotide primer, 2.5 μl of a suitable buffer, 0.4 μl of 1.25 μMdNTP, 2.5 units of Taq DNA polymerase (Stratagene) and deionized waterto a total volume of 25 μl. Mineral oil is overlaid and the PCR isperformed using a programmable thermal cycler.

[0335] The length and temperature of each step of a PCR cycle, as wellas the number of cycles, are adjusted according to the stringencyrequirements in effect. Annealing temperature and timing are determinedboth by the efficiency with which a primer is expected to anneal to atemplate and the degree of mismatch that is to be tolerated. The abilityto optimize the stringency of primer annealing conditions is well withinthe knowledge of one of moderate skill in the art. An annealingtemperature of between 30° C. and 72° C. is used. Initial denaturationof the template molecules normally occurs at between 92° C. and 99° C.for 4 minutes, followed by 20-40 cycles consisting of denaturation(94-99° C. for 15 seconds to 1 minute), annealing (temperaturedetermined as discussed above; 1-2 minutes), and extension (72° C. for 1minute). The final extension step is generally carried out for 4 minutesat 72° C., and may be followed by an indefinite (0-24 hour) step at 4°C.

[0336] Detection methods generally employed in standard PCR techniquesuse a labeled probe with the amplified DNA in a hybridization assay.Preferably, the probe is labeled, e.g., with ³²P, biotin, horseradishperoxidase (HRP), etc., to allow for detection of hybridization.

[0337] Other means of detection include the use of fragment lengthpolymorphism (PCR FLP), hybridization to allele-specific oligonucleotide(ASO) probes (Saiki et al., 1986, Nature 324:163), or direct sequencingvia the dideoxy method (using amplified DNA rather than cloned DNA). Thestandard PCR technique operates (essentially) by replicating a DNAsequence positioned between two primers, providing as the major productof the reaction a DNA sequence of discrete length terminating with theprimer at the 5′ end of each strand. Thus, insertions and deletionsbetween the primers result in product sequences of different lengths,which can be detected by sizing the product in PCR-FLP. In an example ofASO hybridization, the amplified DNA is fixed to a nylon filter (by, forexamples UV irradiation) in a series of “dot blots”, then allowed tohybridize with an oligonucleotide probe labeled with HRP under stringentconditions. After washing, terramethylbenzidine (TMB) and hydrogenperoxide are added: HRP oxidizes the hydrogen peroxide, which in turnoxidizes the TMB to a blue precipitate, indicating a hybridized probe.

[0338] A PCR assay for detecting or measuring a nucleic assay accordingto the invention is described in the section entitled “Methods of Use”.

[0339] 2. Nucleic Acids Comprising RNA

[0340] The present invention also provides a nucleic acid comprisingRNA.

[0341] Nucleic acids comprising RNA can be purified according to methodswell known in the art (Ausubel et al., supra). Total RNA can be isolatedfrom cells and tissues according to methods well known in the art(Ausubel et al., supra) and described below.

[0342] RNA is purified from mammalian tissue according to the followingmethod. Following removal of the tissue of interest, pieces of tissue of≦2 g are cut and quick frozen in liquid nitrogen, to prevent degradationof RNA. Upon the addition of a suitable volume of guanidinium solution(for example 20 ml guanidinium solution per 2 g of tissue), tissuesamples are ground in a tissuemizer with two or three 10-second bursts.To prepare tissue guanidinium solution (1 L) 590.8 g guanidiniumisothiocyanate is dissolved in approximately 400 ml DEPC-treated H₂O. 25ml of 2 M Tris-HCl, pH 7.5 (0.05 M final) and 20 ml Na₂EDTA (0.01 Mfinal) is added, the solution is stirred overnight, the volume isadjusted to 950 ml, and 50 ml 2-ME is added.

[0343] Homogenized tissue samples are subjected to centrifugation for 10min at 12,000×g at 12° C. The resulting supernatant is incubated for 2min at 65° C. in the presence of 0.1 volume of 20% Sarkosyl, layeredover 9 ml of a 5.7M CsCl solution (0.1 g CsCl/ml), and separated bycentrifugation overnight at 113,000×g at 22° C. After careful removal ofthe supernatant, the tube is inverted and drained. The bottom of thetube (containing the RNA pellet) is placed in a 50 ml plastic tube andincubated overnight (or longer) at 4° C. in the presence of 3 ml tissueresuspension buffer (5 mM EDTA, 0.5% (v/v) Sarkosyl, 5% (v/v) 2-ME) toallow complete resuspension of the RNA pellet. The resulting RNAsolution is extracted sequentially with 25:24:1phenol/chloroform/isoamyl alcohol, followed by 24:1 chloroform/isoamylalcohol, precipitated by the addition of 3 M sodium acetate, pH 5.2, and2.5 volumes of 100% ethanol, and resuspended in DEPC water (Chirgwin etal., 1979, Biochemistry, 18: 5294).

[0344] Alternatively, RNA is isolated from mammalian tissue according tothe following single step protocol. The tissue of interest is preparedby homogenization in a glass teflon homogenizer in 1 ml denaturingsolution (4M guanidinium thiosulfate, 25 mM sodium citrate, pH 7.0, 0.1M2-ME, 0.5% (w/v) N-laurylsarkosine) per 100 mg tissue. Followingtransfer of the homogenate to a 5-ml polypropylene tube, 0.1 ml of 2 Msodium acetate, pH 4, 1 ml water-saturated phenol, and 0.2 ml of 49:1chloroform/isoamyl alcohol are added sequentially. The sample is mixedafter the addition of each component, and incubated for 15 min at 0-4°C. after all components have been added. The sample is separated bycentrifugation for 20 min at 10,000×g, 4° C., precipitated by theaddition of 1 ml of 100% isopropanol, incubated for 30 minutes at −20°C. and pelleted by centrifugation for 10 minutes at 10,000×g, 4° C. Theresulting RNA pellet is dissolved in 0.3 ml denaturing solution,transferred to a microfuge tube, precipitated by the addition of 0.3 mlof 100% isopropanol for 30 minutes at −20° C., and centrifuged for 10minutes at 10,000×g at 4° C. The RNA pellet is washed in 70% ethanol,dried, and resuspended in 100-200 μl DEPC-treated water or DEPC-treated0.5% SDS (Chomczynski and Sacchi, 1987, Anal. Biochem., 162: 156).

[0345] Nucleic acids comprising RNA can be produced according to themethod of in vitro transcription.

[0346] The technique of in vitro transcription is well known to those ofskill in the art. Briefly, the gene of interest is inserted into avector containing an SP6, T3 or T7 promoter. The vector is linearizedwith an appropriate restriction enzyme that digests the vector at asingle site located downstream of the coding sequence. Following aphenol/chloroform extraction, the DNA is ethanol precipitated, washed in70% ethanol, dried and resuspended in sterile water. The in vitrotranscription reaction is performed by incubating the linearized DNAwith transcription buffer (200 mM Tris-HCl, pH 8.0, 40 mM MgCl₂, 10 mMspermidine, 250 NaCl [T7 or T3] or 200 mM Tris-HCl, pH 7.5, 30 mM MgCl₂,10 mM spermidine [SP6]), dithiothreitol, RNase inhibitors, each of thefour ribonucleoside triphosphates, and either SP6, T7 or T3 RNApolymerase for 30 min at 37° C. To prepare a radiolabeled polynucleotidecomprising RNA, unlabeled UTP will be omitted and 35S-UTP will beincluded in the reaction mixture. The DNA template is then removed byincubation with DNaseI. Following ethanol precipitation, an aliquot ofthe radiolabeled RNA is counted in a scintillation counter to determinethe cpm/μl (Ausubel et al., supra).

[0347] Alternatively, nucleic acids comprising RNA are prepared bychemical synthesis techniques such as solid phase phosphoramidite(described above).

[0348] 3. Nucleic Acids Comprising Oligonucleotides

[0349] A nucleic acid comprising oligonucleotides can be made by usingoligonucleotide synthesizing machines which are commercially available(described above).

[0350] IV. Cleavage Structure

[0351] The invention provides for a cleavage structure that can becleaved by a nuclease (e.g., a FEN nuclease), and therefore teachesmethods of preparing a cleavage structure. The invention also provides alabeled cleavage structure and methods of preparing a labeled cleavagestructure.

[0352] A probe having a secondary structure that changes upon binding ofthe probe to the target nucleic acid is used to prepare a cleavagestructure according to the invention. A probe according to the inventionhas a secondary structure as defined herein, (including a stem loop, ahairpin, an internal loop, a bulge loop, a branched structure and apseudoknot) or multiple secondary structures, cloverleaf type structuresor any three-dimensional structure, as defined hereinabove. Probesuseful for forming a cleavage structure according to the invention mayalso comprise covalently bound or non-covalently bound subunits (e.g., abi-molecular or multi-molecular probe as defined herein).

[0353] A. Preparation of a Cleavage Structure

[0354] In one embodiment, a cleavage structure according to theinvention is formed by incubating a) an upstream, preferably extendable3′ end, preferably an oligonucleotide primer, b) an oligonucleotideprobe having a secondary structure, as defined herein, that changes uponbinding to a target nucleic acid and comprising a binding moiety,located not more than 5000 nucleotides downstream of the upstream primerand c) an appropriate target nucleic acid wherein the target sequence iscomplementary to both primers and d) a suitable buffer (for exampleSentinel Molecular Beacon PCR core buffer (Catalog #600500) or 10×Pfubuffer available from Stratagene (Catalog #200536), under conditionsthat allow the nucleic acid sequence to hybridize to the oligonucleotideprimers (for example 95° C. for 2-5 minutes followed by cooling tobetween approximately 50-60° C.). The optimal temperature will varydepending on the specific probe(s), primers and polymerases. Inpreferred embodiments of the invention a cleavage structure comprises anoverlapping flap wherein the 3′ end of an upstream oligonucleotidecapable of hybridizing to a target nucleic acid (for example A in FIG.4) is complementary to 1 or more base pair(s) of the downstreamoligonucleotide probe (for example C in FIG. 4) that is annealed to atarget nucleic acid and wherein the 1 base pair overlap is directlydownstream of the point of extension of the single stranded flap.

[0355] According to this embodiment of the 3′ end of the upstreamoligonucleotide primer is extended by the synthetic activity of apolymerase according to the invention such that the newly synthesized 3′end of the upstream oligonucleotide primer partially displaces the 5′end of the downstream oligonucleotide probe. Extension is preferablycarried out in the presence of 1×Sentinel Molecular beacon core bufferor 1×Pfu buffer for 15 seconds at 72° C.

[0356] In another embodiment of the invention, a cleavage structureaccording to the invention can be prepared by incubating a targetnucleic acid with a partially complementary oligonucleotide probe havinga secondary structure, as defined herein, that changes upon binding to atarget nucleic acid and comprising a binding moiety, to a target nucleicacid such that the 3′ complementary region anneals to the target nucleicacid and the non-complementary 5′ region that does not anneal to thetarget nucleic acid forms a 5′ flap. Annealing is preferably carried outunder conditions that allow the nucleic acid sequence to hybridize tothe oligonucleotide primer (for example 95° C. for 2-5 minutes followedby cooling to between approximately 50-60° C.) in the presence asuitable buffer (for example 1×Sentinel Molecular beacon core buffer or1×Pfu buffer.

[0357] In another embodiment of the invention, a cleavage structureaccording to the invention can be prepared by incubating a targetnucleic acid with an upstream primer capable of hybridizing to thetarget nucleic acid and a partially complementary oligonucleotide probehaving a secondary structure, as defined herein, that changes uponbinding to a target nucleic acid and comprising a binding moiety, suchthat the 3′ complementary region anneals to the target nucleic acid andthe non-complementary 5′ region that does not anneal to the targetnucleic acid forms a 5′ flap. Annealing is preferably carried out underconditions that allow the nucleic acid sequence to hybridize to theoligonucleotide primer (for example 95° C. for 2-5 minutes followed bycooling to between approximately 50-60° C.) in the presence a suitablebuffer (for example 1×Sentinel Molecular beacon core buffer (Stratagene)or 1×Pfu buffer (Stratagene).

[0358] B. How to Prepare a Labeled Cleavage Structure

[0359] In the present invention, a label is attached to anoligonucleotide probe having a secondary structure, as defined herein,that changes upon binding to a target nucleic acid and comprising abinding moiety, that comprises a cleavage structure. Thus, the cleavedmononucleotides or small oligonucleotides which are cleaved by theendonuclease activity of the flap-specific nuclease can be detected.

[0360] In one embodiment, a labeled cleavage structure according to theinvention is formed by incubating a) an upstream extendable 3′ end,preferably an oligonucleotide primer, b) a labeled probe having asecondary structure, as defined herein, that changes upon binding to atarget nucleic acid and comprising a binding moiety, located not morethan 5000 nucleotides downstream of the upstream primer and c) anappropriate target nucleic acid wherein the target sequence iscomplementary to the oligonucleotides and d) a suitable buffer (forexample 1×Sentinel Molecular beacon core buffer or 1×Pfu buffer), underconditions that allow the nucleic acid sequence to hybridize to theoligonucleotide primers (for example 95° C. for 2-5 minutes followed bycooling to between approximately 50-60° C.). A cleavage structureaccording to the invention also comprises an overlapping flap whereinthe 3′ end of an upstream oligonucleotide capable of hybridizing to atarget nucleic acid (for example A in FIG. 4) is complementary to 1 basepair of the downstream oligonucleotide probe having a secondarystructure, as defined herein, that changes upon binding to a targetnucleic acid comprising a binding moiety (for example C in FIG. 4) thatis annealed to a target nucleic acid and wherein the 1 base pair overlapis directly downstream of the point of extension of the single strandedflap. The 3′ end of the upstream primer is extended by the syntheticactivity of a polymerase such that the newly synthesized 3′ end of theupstream primer partially displaces the labeled 5′ end of the downstreamprobe. Extension is preferably carried out in the presence of 1×SentinelMolecular beacon core buffer or 1×Pfu buffer for 15 seconds at 72° C.

[0361] In another embodiment, a cleavage structure according to theinvention can be prepared by incubating a target nucleic acid with aprobe having a secondary structure, as defined herein, that changes uponbinding to a target nucleic acid and comprising a binding moiety, andfurther comprising a non-complementary, labeled, 5′ region that does notanneal to the target nucleic acid and forms a 5′ flap, and acomplementary 3′ region that anneals to the target nucleic acid.Annealing is preferably carried out under conditions that allow thenucleic acid sequence to hybridize to the oligonucleotide primer (forexample 95° C. for 2-5 minutes followed by cooling to betweenapproximately 50-60° C.) in the presence a suitable buffer (for example1×Sentinel Molecular beacon core buffer or 1×Pfu buffer).

[0362] In another embodiment, a cleavage structure according to theinvention can be prepared by incubating a target nucleic acid with anupstream primer that is capable of hybridizing to the target nucleicacid and a probe having a secondary structure, as defined herein, thatchanges upon binding to a target nucleic acid and comprising a bindingmoiety, and further comprising a non-complementary, labeled, 5′ regionthat does not anneal to the target nucleic acid and forms a 5′ flap, anda complementary 3′ region that anneals to the target nucleic acid.Annealing is preferably carried out under conditions that allow thenucleic acid sequence to hybridize to the oligonucleotide primer (forexample 95° C. for 2-5 minutes followed by cooling to betweenapproximately 50-60° C.) in the presence a suitable buffer (for example1×Sentinel Molecular beacon core buffer or 1×Pfu buffer).

[0363] Subsequently, any of several strategies may be employed todistinguish the uncleaved labeled nucleic acid from the cleavedfragments thereof. The invention provides for methods for detecting theamount of cleaved, released, nucleic acid fragment that is captured bybinding of a binding moiety or a tag to a capture element, on a solidsupport. In this manner, the present invention permits identification ofthose samples that contain a target nucleic acid.

[0364] The oligonucleotide probe is labeled, as described below, byincorporating moieties detectable by spectroscopic, photochemical,biochemical, immunochemical, enzymatic or chemical means. The method oflinking or conjugating the label to the oligonucleotide probe depends,of course, on the type of label(s) used and the position of the label onthe probe. A probe that is useful according to the invention can belabeled at the 5′ end, the 3′ end or labeled throughout the length ofthe probe.

[0365] A variety of labels that would be appropriate for use in theinvention, as well as methods for their inclusion in the probe, areknown in the art and include, but are not limited to, enzymes (e.g.,alkaline phosphatase and horseradish peroxidase) and enzyme substrates,radioactive atoms, fluorescent dyes, chromophores, chemiluminescentlabels, electrochemiluminescent labels, such as Origen™ (Igen), that mayinteract with each other to enhance, alter, or diminish a signal. Ofcourse, if a labeled molecule is used in a PCR based assay carried outusing a thermal cycler instrument, the label must be able to survive thetemperature cycling required in this automated process.

[0366] Among radioactive atoms, ³³P or, ³²P is preferred. Methods forintroducing ³³P or, ³²P into nucleic acids are known in the art, andinclude, for example, 5′ labeling with a kinase, or random insertion bynick translation. “Specific binding partner” refers to a protein capableof binding a ligand molecule with high specificity, as for example inthe case of an antigen and a monoclonal antibody specific therefor.Other specific binding partners include biotin and avidin orstreptavidin, IgG and protein A, and the numerous receptor-ligandcouples known in the art. The above description is not meant tocategorize the various labels into distinct classes, as the same labelmay serve in several different modes. For example, ¹²⁵I may serve as aradioactive label or as an electron-dense reagent. HRP may serve as anenzyme or as antigen for a monoclonal antibody. Further, one may combinevarious labels for desired effect. For example, one might label a probewith biotin, and detect the presence of the probe with avidin labeledwith 125I, or with an anti-biotin monoclonal antibody labeled with HRP.Other permutations and possibilities will be readily apparent to thoseof ordinary skill in the art and are considered as equivalents withinthe scope of the instant invention.

[0367] Fluorophores for use as labels in constructing labeled probes ofthe invention include rhodamine and derivatives (such as Texas Red),fluorescein and derivatives (such as 5-bromomethyl fluorescein), LuciferYellow, IAEDANS, 7-Me₂N-coumarin-4-acetate,7-OH-4-CH₃-coumarin-3-acetate, 7-NH₂-4-CH₃-coumarin-3-acetate (AMCA),imonobromobimane, pyrene trisulfonates, such as Cascade Blue, andmonobromorimethyl-ammoniobimane. In general, fluorophores with wideStokes shifts are preferred, to allow using fluorimeters with filtersrather than a monochromometer and to increase the efficiency ofdetection.

[0368] Probes labeled with fluorophores can readily be used in nuclease(e.g. FEN-nuclease) mediated cleavage of a cleavage structure comprisinga labeled probe according to the invention. If the label is on the5′-end of the probe, the nuclease (e.g. FEN-nuclease) generated labeledfragment is separated from the intact, hybridized probe by procedureswell known in the art.

[0369] In another embodiment of the invention, detection of thehydrolyzed, labeled probe can be accomplished using, for example,fluorescence polarization, a technique to differentiate between largeand small molecules based on molecular tumbling. Large molecules (i.e.,intact labeled probe) tumble in solution much more slowly than smallmolecules. Upon linkage of a fluorescent moiety to an appropriate siteon the molecule of interest, this fluorescent moiety can be measured(and differentiated) based on molecular tumbling, thus differentiatingbetween intact and digested probe.

[0370] In some situations, one can use two interactive labels (e.g.,FRET or non-FRET pairs) on a single oligonucleotide probe with dueconsideration given for maintaining an appropriate spacing of the labelson the oligonucleotide to permit the separation of the labels duringoligonucleotide probe unfolding (e.g., for example due to a change inthe secondary structure of the probe) or hydrolysis. Preferredinteractive labels useful according to the invention include, but arenot limited to rhodamine and derivatives, fluorescein and derivatives,Texas Red, coumarin and derivatives, crystal violet and include, but arenot limited to DAB CYL, TAMRA and NTB (nitrothiazole blue) in additionto any of the FRET or non-FRET labels described herein.

[0371] The fluorescence of the released label is then compared to thelabel remaining bound to the target. It is not necessary to separate thenuclease (e.g. FEN-nuclease) generated fragment and the probe thatremains bound to the target after cleavage in the presence of nuclease(e.g. FEN-nuclease) if the probe is synthesized with a fluorophore and aquencher that are separated by about 20 nucleotides. Alternatively, thequencher is positioned such that the probe will not fluoresce when nothybridized to the target nucleic acid. Such a dual labeled probe willnot fluoresce when intact or when not hybridized to the target nucleicacid (or in the case of bi- or multimolecular probes, when the probe isnot dissociated) because the light emitted from the dye is quenched bythe quencher. Thus, any fluorescence emitted by an intact probe isconsidered to be background fluorescence. In one embodiment, when alabeled probe is cleaved by a FEN nuclease, dye and quencher areseparated and the released fragment will fluoresce. Alternatively, whena labeled probe is hybridized to a target nucleic acid, the distancebetween the dye and the quencher is increased and the level offluorescence increases. In an embodiment wherein the probe is a bi- ormulti-molecular probe, dissociation of the molecules comprising theprobe results in an increase in fluorescence. The amount of fluorescenceis proportional to the amount of nucleic acid target sequence present ina sample.

[0372] In yet another embodiment, two labeled nucleic acids are used,each complementary to separate regions of separate strands of adouble-stranded target sequence, but not to each other, so that alabeled nucleic acid anneals downstream of each primer. For example, thepresence of two probes can potentially double the intensity of thesignal generated from a single label and may further serve to reduceproduct strand reannealing, as often occurs during PCR amplification.The probes are selected so that the probes bind at positions adjacent(downstream) to the positions at which primers bind.

[0373] One can also use multiple probes in the present invention toachieve other benefits. For instance, one could test for any number ofpathogens in a sample simply by putting as many probes as desired intothe reaction mixture; the probes could each comprise a different labelto facilitate detection.

[0374] One can also achieve allele-specific or species-specificdiscrimination using multiple probes in the present invention, forinstance, by using probes that have different T_(m)s and conducting theannealing/cleavage reaction at a temperature specific for only oneprobe/allele duplex. One can also achieve allele specific discriminationby using only a single probe and examining the types of cleavageproducts generated. In one embodiment of the invention, the probe isdesigned to be exactly complementary, at least in the 5′ terminalregion, to one allele but not to the other allele(s). With respect tothe other allele(s), the probe will be mismatched in the 5′ terminalregion of the probe so that a different cleavage product will begenerated as compared to the cleavage product generated when the probeis hybridized to the exactly complementary allele.

[0375] Although probe sequence can be selected to achieve importantbenefits, one can also realize important advantages by selection ofprobe labels(s) and/or tag as defined herein. The labels may be attachedto the oligonucleotide directly or indirectly by a variety oftechniques. Depending on the precise type of label or tag used, thelabel can be located at the 5′ or 3′ end of the probe, locatedinternally in the probe, or attached to spacer arms of various sizes andcompositions to facilitate signal interactions. Using commerciallyavailable phosphoramidite reagents, one can produce oligomers containingfunctional groups (e.g., thiols or primary amines) at either the 5- orthe 3-terminus via an appropriately protected phosphoramidite, and canlabel them using protocols described in, for example, PCR Protocols: AGuide to Methods and Applications, Innis et al., eds. Academic Press,Ind., 1990.

[0376] Methods for introducing oligonucleotide functionalizing reagentsto introduce one or more sulfhydryl, amino or hydroxyl moieties into theoligonucleotide probe sequence, typically at the 5′ terminus, aredescribed in U.S. Pat. No. 4,914,210. A 5′ phosphate group can beintroduced as a radioisotope by using polynucleotide kinase andgamma-³²P-ATP or gamma-³³P-ATP to provide a reporter group. Biotin canbe added to the 5′ end by reacting an aminothymidine residue, or a6-amino hexyl residue, introduced during synthesis, with anN-hydroxysuccinimide ester of biotin. Labels at the 3′ terminus mayemploy polynucleotide terminal transferase to add the desired moiety,such as for example, cordycepin ³⁵S-dATP, and biotinylated dUTP.

[0377] Oligonucleotide derivatives are also available labels. Forexample, etheno-dA and etheno-A are known fluorescent adeninenucleotides that can be incorporated into a nucleic acid probe.Similarly, etheno-dC or 2-amino purine deoxyriboside is another analogthat could be used in probe synthesis. The probes containing suchnucleotide derivatives may be hydrolyzed to release much more stronglyfluorescent mononucleotides by flap-specific nuclease activity.

[0378] C. Cleaving a Cleavage Structure and Generating a Signal

[0379] A cleavage structure according to the invention can be cleaved bythe methods described in the section above, entitled “Nucleases”.

[0380] D. Detection of Released Labeled Fragments

[0381] Detection or verification of the labeled fragments may beaccomplished by a variety of methods well known in the art and may bedependent on the characteristics of the labeled moiety or moietiescomprising a labeled cleavage structure. Preferably, the releasedlabeled fragments are captured by binding of a binding moiety to acapture element attached to a solid support.

[0382] 1. Capture element

[0383] A capture element, according to the invention can be any moietythat specifically binds (e.g. via hydrogen bonding or via an interactionbetween, for example a nucleic acid binding protein and a nucleic acidbinding site or between complementary nucleic acids) a binding moiety,as a result of attractive forces that exist between the binding moietyand the capture element.

[0384] According to the invention, a binding moiety includes a region ofa probe that binds to a capture element. A capture element according tothe invention can be a nucleic acid sequence that is complementary toand binds to, for example, via hydrogen bonding, a binding moietycomprising a region of a probe that binds to a capture element. Forexample, a binding moiety is a region of a probe comprising the nucleicacid sequence 5′AGCTACTGATGCAGTCACGT3′ and the corresponding captureelement comprises the nucleic acid sequence 5′TCGATGACTACGTCAGTGCA3′.

[0385] The invention also provides for binding moiety-capture element ortag-capture element pairs wherein the binding moiety or tag is a DNAbinding protein and the corresponding capture element is the DNAsequence recognized and bound by the DNA binding protein. The inventionalso provides for binding moiety-capture element or tag-capture elementpairs wherein the capture element is a DNA binding protein and thecorresponding binding moiety or tag is the DNA sequence recognized andbound by the DNA binding protein.

[0386] DNA sequence/DNA binding protein interactions useful according tothe invention include but are not limited to c-myb, AAF, abd-A, Abd-B,ABF-2, ABF1, ACE2, ACF, ADA2, ADA3, Adf-1, Adf-2a, ADR1, AEF-1, AF-2,AFP1, AGIE-BP1, AhR, AIC3, AIC4, AID2, AIIN3, ALF1B, alpha-1, alpha-CPl,alpha-CP2a, alpha-CP2b, alpha-factor, alpha-PAL, alpha2uNF1, alpha2uNF3,alphaA-CRYBP1, alphaH2-alphaH3, alphaMHCBF1, aMEF-2, AML1, AnCF, ANF,ANF-2, Antp, AP-1, AP-2, AP-3, AP-5, APETALA1, APETALA3, AR, ARG RI, ARGRII, Arnt, AS-C T3, AS321, ASF-1, ASH-1, ASH-3b, ASP, AT-13P2, ATBF1-A,ATF, ATF-1, ATF-3, ATF-3deltaZIP, ATF-adelta, ATF-like, Athb-1, Athb-2,Axial, abaA, ABF-1, Ac, ADA-NF1, ADD1, Adf-2b, AF-1, AG, AIC2, AIC5,ALF1A, alpha-CBF, alpha-CP2a, alpha-CP2b, alpha-IRP, alpha2uNF2,alphaH0, AmdR, AMT1, ANF-1, Ap, AP-3, AP-4, APETALA2, aRA, ARG RIII,ARP-1, Ase, ASH-3a, AT-BP1, ATBF1-B, ATF-2, ATF-a, ATF/CREB, Ato, Bfactor, B″, B-Myc, B-TFIID, band I factor, BAP, Bcd, BCFI, Bcl-3,beta-1, BETA1, BETA2, BF-1, BGP1, BmFTZ-F1, BP1, BR-C Z1, BR-C Z2, BR-CZ4, Brachyury, BRF1, Br1A, Brn-3a, Brn-4, Brn-5, BUF1, BUF2, B-Myb,BAF1, BAS1, BCFII, beta-factor, BETA3, BLyF, BP2, BR-C Z3, brahma, byr3,c-ab1, c-Ets-1, c-Ets-2, c-Fos, c-Jun, c-Maf, c-myb, c-Myc, c-Qin,c-Rel, C/EBP, C/EBPalpha, C/EBPbeta, C/EBPdelta, C/EBPepsilon,C/EBPgamma, C1, CAC-binding protein, CACCC-binding factor, Cactus, Cad,CAD1, CAP, CArG box-binding protein, CAUP, CBF, CBP, CBTF, CCAAT-bindingfactor, CCBF, CCF, CCK-1a, CCK-1b, CD28RC, CDC10, Cdc68, CDF, cdk2, CDP,Cdx-1, Cdx-2, Cdx-3, CEBF, CEH-18, CeMyoD, CF1, Cf1a, CF2-I, CF2-II,CF2-III, CFF, CG-1, CHOP-10, Chox-2.7, CIIIB1, Clox, Cnc, CoMP1,core-binding factor, CoS, COUP, COUP-TF, CP1, CP1A, CP1B, CP2, CPBP,CPC1, CPE binding protein CPRF-1, CPRF-2, CPRF-3, CRE-BP1, CRE-BP2,CRE-BP3, CRE-BPa, CreA, CREB, CREB-2, CREBomega, CREMalpha, CREMbeta,CREMdelta, CREMepsilon, CREMgamma, CREMtaualpha, CRF, CSBP-1, CTCF, CTF,CUP2, Cut, Cux, Cx, cyclin A, CYS3, D-MEF2, Da, DAL82, DAP, DAT1, DBF-A,DBF4, DBP, DBSF, dCREB, dDP, dE2F, DEF, Delilah, delta factor,deltaCREB, deltaE1, deltaEF1, deltaMax, DENF, DEP, DF-1, Dfd, dFRA,dioxin receptor, dJRA, D1, DII, D1x, DM-SSRP1, DMLP1, DP-1, Dpn, Dr1,DRTF, DSC1, DSP1, DSXF, DSXM, DTF, E, E1A, E2, E2BP, E2F, E2F-BF, E2F-I,E4, E47, E4BP4, E4F, E4TF2, E7, E74, E75, EBF, EBF1, EBNA, EBP, EBP40,EC, ECF, ECH, EcR, eE-TF, EF-1A, EF-C, EF1, EFgamma, Egr, eH-TF, EIIa,EivF, EKLF, Elf-1, Elg, Elk-1, ELP, Elt-2, EmBP-1, embryo DNA bindingprotein, Emc, EMF, Ems, Emx, En, ENH-binding protein, ENKTF-1,epsilonF1, ER, Erg, Esc, ETF, Eve, Evi, Evx, Exd, Ey, f(alpha-epsilon),F-ACT1, f-EBP, F2F, factor 1-3, factor B1, factor B2, factor delta,factor I, FBF-Al, Fbf1, FKBP59, Fkh, F1bD, Flh, Fli-1, FLV-1, Fos-B,Fra-2, FraI, FRG Y1, FRG Y2, FTS, Ftz, Ftz-F1, G factor, G6 factor,GA-BF, GABP, GADD 153, GAF, GAGA factor, GAL4, GAL80, gamma-factor,gammaCAAT, gammaCAC, gammaOBP, GATA-1, GATA-2, GATA-3, GBF, GC1, GCF,GCF, GCN4, GCR1, GE1, GEBF-I, GF1, GFI, Gfi-1, GFII, GHF-5, GL1, Glass,GLO, GM-PBP-1, GP, GR, GRF-1, Gsb, Gsbn, Gsc, Gt, GT-1, Gtx, H, H16,Hl1TF1, H2Babp1, H2RIIBP, H2TF1, H4TF-1, HAC1, HAP1, Hb, HBLF, HBP-1,HCM1, heat-induced factor, HEB, HEF-1B, HEF-1T, HEF-4C, HEN1, HES-1,HIF-1, HiNF-A, HIP1, HIV-EP2, Hlf, HMBI, HNF-1, HNF-3, Hox11, HOXA1,HOXA10, HOXA10PL2, HOXA11, HOXA2, HOXA3, HOXA4, HOXA5, HOXA7, HOXA9,HOXB1, HOXB3, HOXB4, HOXB5, HOXB6, HOXB7, HOXB8, HOXB9, HOXC5, HOXC6,HOXC8, HOXD1, HOXD10, HOXD11, HOXD12, HOXD13, HOXD4, HOXD8, HOXD9, HP1site factor, Hp55, Hp65, HrpF, HSE-binding protein, HSF1, HSF2, HSF24,HSF3, HSF30, HSF8, hsp56, Hsp90, HST, HSTF, I-POU, IBF, IBP-1, ICER,ICP4, ICSBP, Id1, Id2, Id3, Id4, IE1, EBP1, IEFga, IF1, IF2, IFNEX,IgPE-1, IK-1, IkappaB, Il-1 RF, IL-6 RE-BP, Il-6 RF, ILF, ILRF-A, IME1,INO2, INSAF, IPF1, IRBP, IRE-ABP, IREBF-1, IRF-1, ISGF-1, Isl-1, ISRF,ITF, IUF-1, Ixr1, JRF, Jun-D, JunB, JunD, K-2, kappay factor, kBF-A,KBF1, KBF2, KBP-1, KER-1, Ker1, KN1, Kni, Knox3, Kr, kreisler, KRF-1,Krox-20, Krox-24, Ku autoantigen, KUP, Lab, LAC9, LBP, Lc, LCR-F1,LEF-1, LEF-1S, LEU3, LF-A1, LF-B1, LF-C, LF-H3beta, LH-2, Lim-1, Lim-3,lin-11, lin-31, lin-32, LIP, LIT-1, LKLF, Lmx-1, LRF-1, LSF, LSIRF-2,LVa, LVb-binding factor, LVc, LyF-1, Lyl-1, M factor, M-Twist, M1, m3,Mab-18, MAC1, Mad, MAF, MafB, MafF, MafG, MafK, Mal63, MAPF1, MAPF2,MASH-1, MASH-2, mat-Mc, mat-Pc, MATa1, MATalpha1, MATalpha2, MATH-1,MATH-2, Max1, MAZ, MBF-1, MBP-1, MBP-2, MCBF, MCM1, MDBP, MEB-1, Mec-3,MECA, mediating factor, MEF-2, MEF-2C, MEF-2D, MEF1, MEP-1, Mesol, MF3,Mi, MIF, MIG1, MLP, MNB1a, MNF1, MOK-2, MP4, MPBF, MR, MRF4, MSN2, MSN4,Msx-1, Msx-2, MTF-1, mtTF1, muEBP-B, muEBP-C2, MUF1, MUF2, Mxi1, Myef-2,Myf-3, Myf-4, Myf-5, Myf-6, Myn, MyoD, myogenin, MZF-1, N-Myc, N-Oct-2,N-Oct-3, N-Oct-4, N-Oct-5, Nau, NBF, NC1, NeP1, Net, NeuroD, neurogenin,NF III-a, NF-1, NF-4FA, NF-AT, NF-BA1, NF-CLE0a, NF-D, NF-E, NF-E1b,NF-E2, NF-EM5, NF-GMa, NF-H1, NF-IL-2A, NF-InsE1, NF-kappaB, NF-lambda2,NF-MHCIIA, NF-muE1, NF-muNR, NF-S, NF-TNF, NF-U1, NF-W1, NF-X, NF-Y,NF-Zc, NFalphal, NFAT-1, NFbetaA, NFdeltaE3A, NFdeltaE4A, NFe, NFE-6,NFH3-1, NFH3-2, NFH3-3, NFH3-4, NGFI-B, NGFI-C, NHP, Nil-2-a, NIP, NIT2,Nkx-2.5, NLS1, NMH7, NP-III, NP-IV, NP-TCII, NP-Va, NRDI, NRF-1, NRF-2,Nrf1, Nrf2, NRL, NRSF form 1, NTF, NUC-1, Nur77, OBF, OBP, OCA-B, OCSTF,Oct-1, Oct-10, Oct-11, Oct-2, Oct-2.1, Oct-2.3, Oct-4, Oct-5, Oct-6,Oct-7, Oct-8, Oct-9, Oct-B2, Oct-R, Octa-factor, octamer-binding factor,Odd, Olf-1, Opaque-2, Otd, Otx1, Otx2, Ovo, P, P1, p107, p130, p28modulator, p300, p38erg, p40x, p45, p49erg, p53, p55, p55erg, p58,p65delta, p67, PAB1, PacC, Pap1, Paraxis, Pax-1, Pax-2, Pax-3, Pax-5,Pax-6, Pax-7, Pax-8, Pb, Pbx-1a, Pbx-1b, PC, PC2, PC4, PC5, Pcr1, PCRE1,PCT1, PDM-1, PDM-2, PEA1, PEB1, PEBP2, PEBP5, Pep-1, PF1, PGA4, PHD1,PHO2, PHO4, PHO80, Phox-2, Pit-1, PO-B, pointedP1, Pou2, PPAR, PPUR,PPYR, PR, PR A, Prd, PrDI-BF1, PREB, Prh proein a, protein b, proteinc,protein d, PRP, PSE1, PTF, Pu box binding factor, PU.1, PUB 1, PuF,PUF-I, Pur factor, PUT3, pX, qa-1F, QBP, R, R1, R2, RAd-1, RAF, RAP1,RAR, Rb, RBP-Jkappa, RBP60, RC1, RC2, REB1, Re1A, Re1B, repressor ofCAR1 expression, REX-1, RF-Y, RF1, RFX, RGM1, RIM1, RLM1, RME1, Ro,RORalpha, Rox1, RPF1, RPGalpha, RREB-1, RRF1, RSRFC4, runt, RVF,RXR-alpha, RXR-beta, RXR-beta2, RXR-gamma, S-CREM, S-CREMbeta, S8,SAP-1a, SAP1, SBF, Sc, SCBPalpha, SCD1/BP, SCM-inducible factor, Scr,Sd, Sdc-1, SEF-1, SF-1, SF-2, SF-3, SF-A, SGCI, SGF-1, SGF-2, SGF-3,SGF-4, SIF, SIII, Sim, SIN1, Skn-1, SKO1, Slp1, Sn, SNP1, SNF5, SNAPC43,Sox-18, Sox-2, Sox-4, Sox-5, Sox-9, Sox-LZ, Sp1, spE2F, Sph factor,Spi-B, Sprm-1, SRB10, SREBP, SRF, SRY, SSDBP-1, ssDBP-2, SSRP1, STAF-50,STAT, STATI, STAT2, STAT3, STAT4, STAT5, STAT6, STC, STD1, Ste11, Ste12,Ste4, STM, Su(f), SUM-1, SWI1, SWI4, SWI5, SWI6, SWP, T-Ag, t-Pou2, T3R,TAB, all TAFs including subunits, Tal-1, TAR factor, tat, Tax, TBF1,TBP, TCF, TDEF, TEA1, TEC1, TEF, tel, Tf-LFl, TFE3, all TFII relatedproteins, TBA1a, TGGCA-binding protein, TGT3, Th1, TIF1, TIN-1, TIP,T11, TMF, TR2, Tra-1, TRAP, TREB-1, TREB-2, TREB-3, TREF1, TREF2, Tsh,TTF-1, TTF-2, Ttk69k, TTP, Ttx, TUBF, Twi, TxREBP, TyBF, UBP-1, Ubx,UCRB, UCRF-L, UF1-H3beta, UFA, UFB, UHF-1, UME6, Unc-86, URF, URSF,URTF, USF, USF2, v-ErbA, v-Ets, v-Fos, v-Jun, v-Maf, v-Myb, v-Myc,v-Qin, v-Rel, Vab-3, vaccinia virus DNA-binding protein, Vav, VBP, VDR,VETF, vHNF-1, VITF, Vmw65, Vp1, Vp16, Whn, WT1, X-box binding protein,X-Twist, X2BP, XBP-1, XBP-2, XBP-3, XFI, XF2, XFD-1, XFD-3, xMEF-2,XPF-1, XrpFI, XW, XX, yan, YB-1, YEB3, YEBP, Yi, YPF1, YY1, ZAP, ZEM1,ZEM2/3, Zen-1, Zen-2, Zeste, ZF1, ZF2, Zfh-1, Zfh-2, Zfp-35, ZID,Zmhoxla, Zta and all related characterized and uncharacterized homologsand family members related to these DNA binding proteins or activities,and the DNA sequence recognized by the above-recited DNA bindingproteins. Methods of identifying a DNA sequence recognized by a DNAbinding protein are known in the art (see for example, U.S. Pat. No.6,139,833).

[0387] The invention also contemplates DNA sequence/DNA binding proteininteractions including but not limited to the tetracycline (tet)repressor, beta.-galactosidase (lac repressor), the tryptophan (trp)repressor, the lambda specific repressor protein, CRO, and thecatabolite activator protein, CAP and the DNA sequence recognized byeach of these DNA binding proteins and known in the art. DNA/DNA bindingprotein interactions useful according to the invention also includerestriction enzymes and the corresponding restriction sites, preferablyunder conditions wherein the nuclease activity of the restriction enzymeis suppressed (U.S. Pat No. 5,985,550, incorporated herein byreference).

[0388] Other DNA:Protein interactions useful according to the inventioninclude (i) the DNA protein interactions listed in Tables 1 and 2, and(ii) bacterial, yeast, and phage systems such as lambda OL-OR/CrO (U.S.Pat. No. 5,726,014, incorporated herein by reference). Any paircomprising a protein that binds to a specific recognition sequence andthe cognate recognition sequence may be useful in the present invention.TABLE 1 DNA-BINDING SEQUENCES Test sequence DNA-binding Protein EBVorigin of replication EBNA HSV origin of replication UL9 VZV origin ofreplication UL9-like HPV origin of replication E2 Interleukin 2 enhancerNFAT-1 HIV LTR NFAT-1 NFkB HBV enhancer HNF-1 Fibrogen promoter HNF-1

[0389] TABLE 2 Name DNA Sequence Recognized* Bacteria lac repressorAATTGTGAGCGGATAACAATT TTAACACTCGCCTATTGTTAA CAP TGTGAGTTAGCTCACTACACTCAATCGAGTGA lambda repressor TATCACCGCCAGAGGTA ATAGTGGCGGTCTCCATYeast GAL4 CGGAGGACTGTCCTCCG GCCTCCTCACAGGAGGC MAT α2 CATGTAATTGTACATTAA GCN4 ATGACTCAT TACTGAGTA Drosophila Krüppel AACGGGTTAATTGCCCAATT bicoid GGGATTAGA CCCTAATCT Mammals Sp1 GGGCGG CCCGCC Oct-1ATGCAAAT TACGTTTA GATA-1 TGATAG ACTATC

[0390] Methods of performing a reaction wherein specific binding occursbetween a capture element, as defined herein and a binding moiety, asdefined herein, are well known in the art, see for example, Sambrook etal., supra; Ausubel et al., supra). A capture element, according to theinvention can also be any moiety that specifically binds (e.g. viacovalent or hydrogen bonding or electrostatic attraction or via aninteraction between, for example a protein and a ligand, an antibody andan antigen, protein subunits, a nucleic acid binding protein and anucleic acid binding site) a binding moiety or a tag, as a result ofattractive forces that exist between the binding moiety or tag and thecapture element. Methods of performing a reaction wherein specificbinding occurs between a capture element, as defined herein and a tag,as defined herein, are well known in the art, see for example, Sambrooket al., supra; Ausubel et al., supra). Specific binding only occurs whenthe secondary structure of the probe comprising the binding moiety has“changed”, as defined herein. Capture elements useful according to theinvention include but are not limited to a nucleic acid binding proteinor a nucleotide sequence, biotin, streptavidin, a hapten, a protein, anucleotide sequence or a chemically reactive moiety.

[0391] In one embodiment of the invention, the reaction products,including the released labeled fragments, are subjected to sizeanalysis. Methods for determining the size of a labeled fragment areknown in the art and include, for example, gel electrophoresis,sedimentation in gradients, gel exclusion chromatography, massspectroscopy, and homochromatography.

[0392] 2. Solid Substrate

[0393] A solid substrate according to the invention is any surface towhich a molecule (e.g., capture element) can be irreversibly bound,including but not limited to membranes, magnetic beads, tissue cultureplates, silica based matrices, membrane based matrices, beads comprisingsurfaces including but not limited to styrene, latex or silica basedmaterials and other polymers for example cellulose acetate, teflon,polyvinylidene difluoride, nylon, nitrocellulose, polyester, carbonate,polysulphone, metals, zeolites, paper, alumina, glass, polypropyle,polyvinyl chloride, polyvinylidene chloride, polytetrafluorethylene,polyethylene, polyamides, plastic, filter paper, dextran, germanium,silicon, (poly)tetrafluorethylene, gallium arsenide, gallium phosphide,silicon oxide, silicon nitrate and combinations thereof.

[0394] Useful solid substrates according to the invention are alsodescribed in Sambrook et al., supra, Ausubel et al., supra, U.S. Pat.Nos. 5,427,779, 5,512,439, 5,589,586, 5,716,854 and 6,087,102, Southernet al., 1999, Nature Genetics Supplement, 21:5 and Joos et al., 1997,Analytical Biochemistry, 247:96.

[0395] Methods of attaching a capture element to a solid support areknown in the art and are described in Sambrook et al., supra, Ausubel etal., supra, U.S. Pat. Nos. 5,427,779, 5,512,439, 5,589,586, 5,716,854and 6,087,102 and in Southern et al., supra and Joos et al., supra.Methods of immobilizing a nucleic acid sequence on a solid support arealso provided by the manufacturers of the solid support, e.g., formembranes: Pall Corporation, Schleicher & Schuell, for magnetic beads;Dyal, for culture plates; Costar, Nalgenunc, and for other supportsuseful according to the invention, CPG, Inc.

[0396] The amount of released labeled fragment that is bound to acapture element attached to a solid support can be measured while thelabeled fragment remains bound to the capture element or after releaseof the labeled fragment from the capture element. Release of a labeledfragment from a capture element is carried out by incubating labeledfragment-capture element complexes in the presence of an excess amountof a competing, unlabeled fragment or by the addition of a buffer thatinhibits binding of the labeled fragment to the capture element, forexample as a result of salt concentration or pH.

[0397] During or after amplification, separation of the released labeledfragments from, for example, a PCR mixture can be accomplished by, forexample, contacting the PCR with a solid phase extractant (SPE). Forexample, materials having an ability to bind nucleic acids on the basisof size, charge, or interaction with the nucleic acid bases can be addedto the PCR mixture, under conditions where labeled, uncleaved nucleicacids are bound and short, labeled fragments are not. Such SPE materialsinclude ion exchange resins or beads, such as the commercially availablebinding particles Nensorb (DuPont Chemical Co.), Nucleogen (The NestGroup), PEI, BakerBond™ PEI, Amicon PAE 1,000, Selectacel™ PEI, BoronateSPE with a 3′-ribose probe, SPE containing sequences complementary tothe 3′-end of the probe, and hydroxylapatite. In a specific embodiment,if a dual labeled oligonucleotide comprising a 3′ biotin label separatedfrom a 5′ label by a nuclease susceptible cleavage site is employed asthe signal means, the reaction mixture, for example a PCR amplifiedmixture can be contacted with materials containing a specific bindingelement such as avidin or streptavidin, or an antibody or monoclonalantibody to biotin, bound to a solid support such as beads andparticles, including magnetic particles.

[0398] Following the step in which a reaction mixture, for example a PCRmixture has been contacted with an SPE, the SPE material can be removedby filtration, sedimentation, or magnetic attraction, leaving thelabeled fragments free of uncleaved labeled oligonucleotides andavailable for detection.

[0399] 3. Binding Moieties

[0400] A binding moiety according to the invention refers to a region ofa probe that is released upon cleavage of the probe by a nuclease andbinds specifically (via hydrogen binding with a complementary nucleicacid or via an interaction with a binding protein) to a capture elementas a result of attractive forces that exist between the binding moietyand the capture element, and wherein specific binding between thebinding moiety and the capture element only occurs when the secondarystructure of the probe has “changed”, as defined herein.

[0401] A “tag” refers to a moiety that is operatively linked to the 5′end of a probe (for example R in FIG. 1) and specifically binds to acapture element as a result of attractive forces that exist between thetag and the capture element, and wherein specific binding between thetag and the capture element only occurs when the secondary structure ofthe probe has changed (for example, such that the tag is accessible to acapture element). “Specifically binds” as it refers to a “tag” and acapture element means via covalent or hydrogen bonding or electrostaticattraction or via an interaction between, for example a protein and aligand, an antibody and an antigen, protein subunits, or a nucleic acidbinding protein and a nucleic acid binding site. Second binding moietiesinclude but are not limited to biotin, streptavidin, a hapten, aprotein, or a chemically reactive moiety.

[0402] According to the invention, a binding moiety includes a region ofa probe that binds to a capture element. A capture element according tothe invention can be a nucleic acid sequence that is complementary toand binds to, for example, via hydrogen bonding, a binding moietycomprising a region of a probe that binds to a capture element. Forexample, a binding moiety is a region of a probe comprising the nucleicacid sequence 5′AGCTACTGATGCAGTCACGT3′ and the corresponding captureelement comprises the nucleic acid sequence 5′TCGATGACTACGTCAGTGCA3′.

[0403] The invention also provides for binding moiety-capture element ortag-capture element pairs wherein the binding moiety or tag is a DNAbinding protein and the corresponding capture element is the DNAsequence recognized and bound by the DNA binding protein. The inventionalso provides for binding moiety-capture element or tag-capture elementpairs wherein the capture element is a DNA binding protein and thecorresponding binding moiety or tag is the DNA sequence recognized andbound by the DNA binding protein.

[0404] DNA binding sequence/DNA binding protein interactions usefulaccording to the invention are described above in the section entitled,“Detection of Released Labeled Fragments”.

[0405] Methods of incorporating a tag, as defined herein, into a nucleicacid (e.g., a probe according to the invention) are well known in theart and are described in Ausubel et al., supra, Sambrook et al., supra,and U.S. Pat. Nos. 5,716,854 and 6,087,102.

[0406] IV. Determining the Stability of the Secondary Structure of aProbe

[0407] A. Melting Temperature Assay

[0408] A melting temperature assay, takes advantage of the differentabsorption properties of double stranded and single stranded DNA, thatis, double stranded DNA (the double stranded DNA being that portion of anucleic acid sequence that has folded back on itself to generate anantiparallel duplex structure wherein complementary sequences (basepairs) are associated via hydrogen bonding) absorbs less light thansingle stranded DNA at a wavelength of 260 nm, as determined byspectrophotometric measurement.

[0409] The denaturation of DNA occurs over a narrow temperature rangeand results in striking changes in many of the physical properties ofDNA. A particularly useful change occurs in optical density. Theheterocyclic rings of nucleotides adsorb light strongly in theultraviolet range (with a maximum close to 260 mn that is characteristicfor each base). However, the adsorption of DNA is approximately 40% lessthan would be displayed by a mixture of free nucleotides of the samecomposition. This effect is called hyperchromism and results frominteractions between the electron systems of the bases, made possible bytheir stacking in the parallel array of the double helix. Any departurefrom the duplex state is immediately reflected by a decline in thiseffect (that is, by an increase in optical density toward the valuecharacteristic of free bases (FIG. 12a). The denaturation of doublestranded DNA can therefore be followed by this hyperchromicity (FIGS.12b and 12 c).

[0410] The midpoint of the temperature range over which the strands ofDNA separate is called the melting temperature, denoted T_(m). Anexample of a melting curve determined by change in optical absorbance isshown in FIG. 12c. The curve always takes the same form, but itsabsolute position on the temperature scale (that is, its T_(m)) isinfluenced by both the base composition of the DNA and the conditionsemployed for denaturation.

[0411] The melting temperature of a DNA molecule depends markedly on itsbase composition. DNA molecules rich in GC base pairs have a higher Tmthan those having an abundance of AT base pairs (FIG. 13b). The Tm ofDNA from many species varies linearly with GC content, rising from 77°to 100° C. as the fraction of GC pairs increases from 20% to 78%. Thatis, the dependence of T_(m) on base composition is linear, increasingabout 0.4° C. for every percent increase in G-C content. GC base pairsare more stable than AT pairs because their bases are held together bythree hydrogen bonds rather than by two. In addition, adjacent GC basepairs interact more strongly with one another than do adjacent AT basepairs. Hence, the AT-rich regions of DNA are the first to melt.

[0412] A major effect on T_(m) is exerted by the ionic strength of thesolution. The T_(m) increases 16.6° C. for every tenfold increase inmonovalent cation concentration. The most commonly used condition is toperform manipulations of DNA in 0.12 M phosphate buffer, which providesa monovalent Na+ concentration of 0.1 8M, and a T_(m) of the order of90° C.

[0413] The T_(m) can be greatly varied by performing the reaction in thepresence of reagents, such as formamide, that destabilize hydrogenbonds. This allows the T_(m) to be reduced to as low as 40° C. with theadvantage that the DNA does not suffer damage (such as strand breakage)that can result from exposure to high temperatures. (Stryer,Biochemistry, 1998, 3^(rd) Edition, W. H. Freeman and Co., pp.81-82 andLewin, Genes II, 1985, John Wiley & Sons, p.63-64).

[0414] The stability of the secondary structure of the probe accordingto the invention is determined in a melting temperature assay asfollows.

[0415] A standard curve for the probe (for example FIG. 12c), whereinabsorbance is plotted versus temperature, is prepared by incubating asample comprising from about 0.2 μg/ml to 100 μg/ml of the probe in abuffer which allows for denaturing and reannealing of the probe atvarious temperatures for a time sufficient to permit denaturing andreannealing of the probe and measuring the absorbance of a sample in aquartz cuvette (with a pathlength appropriate for the spectrophotometerbeing used, e.g., 1-cm), in a spectrophotometer over a range oftemperatures wherein the lower temperature limit of the range is atleast 50° C. below, and the upper temperature limit of the range is atleast 50° C. above the Tm or predicted Tm of the probe. The Tm of theprobe is predicted based on the base pair composition according tomethods well known in the art (see, Sambrook, supra; Ausubel, supra).Standard curves are generated and compared, using a variety of buffers(e.g., 1×TNE buffer (10×-0.1M Tris base, 10 mM EDTA, 2.0 M NaCl , pH7.4), FEN nuclease buffer, described herein, 1×Cloned Pfu buffer,described herein, 1×Sentinel Molecular beacon buffer, described herein)including a buffer that is possible and preferentially optimal for theparticular nuclease to be employed in the cleavage reaction. The pH ofthe buffer will be monitored as the temperature increases, and adjustedas is needed.

[0416] The assay is performed in a single-beam ultraviolet to visiblerange (UV-VIS) spectrophotometer. Preferably, the assay is performed ina double-beam spectrophotometer to simplify measurements byautomatically comparing the cuvette holding the sample solution to areference cuvette (matched cuvette) that contains the blank. The blankis an equal volume of sample buffer.

[0417] The temperature of the spectrophotometer can be controlled suchthat the absorbance of the sample is measured at specific temperatures.Spectrophotometers useful according to the invention include but are notlimited to the Beckman Coulter DU® 600/7000 Spectrophotometers incombination with the MicroTm Analysis Accessory (Beckman Coulter, Inc.,Columbia, Md.).

[0418] The stability of the secondary structure of a probe at aparticular temperature and in a buffer that is possible andpreferentially optimal for the nuclease to be employed in the cleavagereaction of the probe, is determined by measuring the absorbance of theprobe at a particular temperature, as above, and determining if thevalue of the absorbance is less than the absorbance at the Tm, asdetermined from the standard curve, wherein the standard curve isgenerated using either the same buffer as used at the test temperature,or a buffer known to produce a comparable standard curve, as describedabove. The secondary structure of the probe is “stable” in a meltingtemperature assay, at a temperature that is at or below the temperatureof the cleavage reaction (i.e., at which cleavage is performed) if thelevel of light absorbance at the temperature at or below the temperatureof the cleavage reaction is less (i.e., at least 5%, preferably 20% andmost preferably 25% or more) than the level of light absorbance at atemperature that is equal to the Tm of the probe (see FIGS. 12c and 12d).

[0419] B. FRET

[0420] A FRET assay is useful in the invention for two purposes. Thefirst is to determine whether the secondary structure of a probe is“stable” as defined herein. The second is to determine whether thesecondary structure of the probe has undergone a “change” upon bindingof the probe to the target nucleic acid.

[0421] “FRET” is a distance-dependent interaction between the electronicexcited states of two dye molecules in which excitation is transferredfrom a donor molecule to an acceptor molecule. FRET is caused by achange in the distance separating a fluorescent donor group from aninteracting resonance energy acceptor, either another fluorophore, achromophore, or a quencher. Combinations of donor and acceptor moietiesare known as “FRET pairs”. Efficient FRET interactions require that theabsorption and emission spectra of the dye pairs have a high degree ofoverlap.

[0422] In most embodiments, the donor and acceptor dyes for FRET aredifferent, in which case FRET can be detected by the appearance ofsensitized fluorescence of the acceptor and/or by quenching of donorfluorescence. When the donor and acceptor are the same, FRET is detectedby the resulting fluorescence depolarization. FRET is dependent on theinverse sixth power of the intermolecular separation (Stryer et al.,1978, Ann. Rev. Biochem., 47:819; Selvin, 1995, Methods Enzymol.,246:300).

[0423] As used herein, the term “donor” refers to a fluorophore whichabsorbs at a first wavelength and emits at a second, longer wavelength.The term “acceptor” refers to a fluorophore, chromophore or quencherwith an absorption spectrum which overlaps the donor's emission spectrumand is able to absorb some or most of the emitted energy from the donorwhen it is near the donor group (typically between 1-100 nm). If theacceptor is a fluorophore capable of exhibiting FRET, it then re-emitsat a third, still longer wavelength; if it is a chromophore or quencher,then it releases the energy absorbed from the donor without emitting aphoton. Although the acceptor's absorption spectrum overlaps the donor'semission spectrum when the two groups are in proximity, this need not bethe case for the spectra of the molecules when free in solution.Acceptors thus include fluorophores, chromophores or quenchers whichexhibit either FRET or quenching when placed in proximity, on a probeaccording to the invention, to the donor due to the presence of a probesecondary structure that changes upon binding of the probe to the targetnucleic acid, as defined herein. Acceptors do not include fluorophores,chromophores or quenchers that exhibit FRET or quenching a) attemperatures equal to or greater than the Tm (e.g. more than 5° abovethe Tm, for example 6°, 10°, 25°, 50° or more above the Tm) or b) in thepresence of a target nucleic acid.

[0424] Reference herein to “fluorescence” or “fluorescent groups” or“fluorophores” include luminescence, luminescent groups and suitablechromophores, respectively. Suitable luminescent probes include, but arenot limited to, the luminescent ions of europium and terbium introducedas lanthium chelates (Heyduk & Heyduk, 1997). The lanthanide ions arealso good donors for energy transfer to fluorescent groups (Selvin1995). Luminescent groups containing lanthanide ions can be incorporatedinto nucleic acids utilizing an ‘open cage’ chelator phosphoramidite.

[0425] As used herein, the term “quenching” refers to the transfer ofenergy from donor to acceptor which is associated with a reduction ofthe intensity of the fluorescence exhibited by the donor.

[0426] The donor and acceptor groups may independently be selected fromsuitable fluorescent groups, chromophores and quenching groups. Donorsand acceptors useful according to the invention include but are notlimited to: 5-FAM (also called 5-carboxyfluorescein; also calledSpiro(isobenzofuran-1(3H), 9′-(9H)xanthene)-5-carboxylicacid,3′,6′-dihydroxy-3-oxo-6-carboxyfluorescein);5-Hexachloro-Fluorescein([4,7,2′,4′,5′,7′-hexachloro-(3′,6′-dipivaloyl-fluoresceinyl)-6-carboxylicacid]); 6-Hexachloro-Fluorescein([4,7,2′,4′,5′,7′-hexachloro-(3′,6′-dipivaloylfluoresceinyl)-5-carboxylicacid]); 5-Tetrachloro-Fluorescein([4,7,2′,7′-tetrachloro-(3′,6′-dipivaloylfluoresceinyl)-5-carboxylicacid]); 6-Tetrachloro-Fluorescein([4,7,2′,7′-tetrachloro-(3′,6′-dipivaloylfluoresceinyl)-6-carboxylicacid]); 5-TAMRA (5-carboxytetramethylrhodamine; Xanthylium,9-(2,4-dicarboxyphenyl)-3,6-bis(dimethyl-amino); 6-TAMRA(6-carboxytetramethylrhodamine; Xanthylium, 9-(2,5-dicarboxyphenyl)-3,6-bis(dimethylamino); EDANS(5-((2-aminoethyl)amino)naphthalene-1-sulfonic acid); 1,5-IAEDANS(5-((((2-iodoacetyl)amino)ethyl)amino)naphthalene-1-sulfonic acid);DABCYL (4-((4-(dimethylamino)phenyl)azo)benzoic acid) Cy5(Indodicarbocyanine-5) Cy3 (Indo-dicarbocyanine-3); and BODIPY FL(2,6-dibromo-4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-proprionicacid), as well as suitable derivatives thereof.

[0427] In certain embodiments of the invention, a probe may also belabeled with two chromophores, and a change in the absorption spectra ofthe label pair is used as a detection signal, as an alternative tomeasuring a change in fluorescence.

[0428] In the method of the invention, fluorescence intensity of theprobe is measured at one or more wavelengths with a fluorescencespectrophotometer or microtitre plate reader, according to methods knownin the art.

[0429] C. Fluorescence Quenching Assay

[0430] A fluorescence quenching assay is useful in the invention for twopurposes. The first is to determine whether the secondary structure of aprobe is “stable” as defined herein. The second is to determine whetherthe secondary structure of the probe has undergone a “change” uponbinding of the probe to the target nucleic acid.

[0431] A probe according to the invention is labeled with a pair ofinteractive labels (e.g., a FRET or non-FRET pair) wherein one member ofthe pair is a fluorophore and the other member of the pair is aquencher. For example, a probe according to the invention is labeledwith a fluorophore and a quencher and fluorescence is measured in theabsence of a target nucleic acid, over a range of temperatures, e.g.,wherein the lower temperature limit of the range is at least 50° Celsiusbelow, and the upper temperature limit of the range is at least 50°Celsius above the Tm or the predicted Tm of the probe.

[0432] D. Stability

[0433] The “stability” of the secondary structure of a probe accordingto the invention is determined as follows. A probe is labeled with apair of interactive labels (for example, tetramethylrhodamine andDABCYL, or any of the interactive labels (either FRET or non-FRET pairs)described herein according to methods well known in the art (for exampleas described in Glazer and Mathies, 1997, Curr. Opin. Biotechnol., 8:94;Ju et al., 1995, Analytical Biochem., 231:131)). The location of theinteractive labels on the probe is such that the labels are separatedwhen the secondary structure of the probe changes following binding ofthe probe to the target nucleic acid.

[0434] A standard curve for the probe (for example FIG. 12e), whereinfluorescence is plotted versus temperature, is prepared by incubating asample comprising typically 125 nM probe in 1×Melting Buffer (20 mMTris-HCl, pH 8.0, 1 mM MgCl₂) or alternatively, in 5 mM Tris-HCl, pH8.0, 0.1 mM EDTA, or other appropriate buffers for a time that issufficient to permit denaturing and reannealing of the probe (typicallythe standard curve is generated using a fluorometer or spectrometer thatundergoes a 1° C. per minute change, and measuring the fluorescence in afluorometer or scanning fluorescence spectrophotometer over a range oftemperatures wherein the lower temperature limit of the range is atleast 50° C. below, and the upper temperature limit of the range is atleast 50° C. above the Tm or predicted Tm of the probe. The Tm of theprobe is predicted based on the base pair composition according tomethods well known in the art (see, Sambrook, supra; Ausubel, supra).

[0435] Standard curves are generated and compared, using a variety ofbuffers (e.g., 1×TNE buffer (10×-0.1M Tris base, 10 mM EDTA, 2.0 M NaCl, pH 7.4), FEN nuclease buffer, described herein, 1×Cloned Pfu buffer,described herein, 1×Sentinel Molecular beacon buffer, described herein)including a buffer that is possible and preferentially optimal for theparticular nuclease to be employed in the cleavage reaction. The pH ofthe buffer will be monitored as the temperature increases, and adjustedas is needed.

[0436] The temperature of the fluorometer or spectrophotometer can becontrolled such that the fluorescence of the sample is measured atspecific temperatures. Fluorescence can be measured for example with aPerkin-Elmer LS50B Luminescence Spectrometer in combination with atemperature regulatable water bath (e.g., for example available fromFisher Scientific).

[0437] The stability of the secondary structure of a probe at aparticular temperature is determined by measuring the fluorescence ofthe probe at a particular temperature, as above, and determining if thevalue of the fluorescence is less than the fluorescence at the Tm, asdetermined from the standard curve. The secondary structure of the probeis “stable” in a FRET assay, at a temperature that is at or below thetemperature of the cleavage reaction (i.e., at which cleavage isperformed) if the level of fluorescence at the temperature at or belowthe temperature of the cleavage reaction is altered (i.e., at least 5%,preferably 20% and most preferably 25% more or less than) the level offluorescence at a temperature that is equal to the Tm of the probe. Thesecondary structure of the probe is “stable” in a fluorescence quenchingassay, at a temperature that is at or below the temperature of thecleavage reaction (i.e., at which cleavage is performed) if the level offluorescence at the temperature at or below the temperature of thecleavage reaction is less (i.e., at least 5%, preferably 20% and mostpreferably 25% more or less than) the level of fluorescence at atemperature that is equal to the Tm of the probe(see FIGS. 12f and 12g).

[0438] Alternatively, the stability of the secondary structure of theprobe is determined by modifying the method of Gelfand et al. (1999,Proc. Natl. Acad. Sci. USA, 96:6113), incorporated herein by reference,to determine the fluorescence of a probe labeled with a pair ofinteractive labels over a range of temperatures, as describedhereinabove.

[0439] V. Detecting a Secondary Structure

[0440] A secondary structure according to the invention is detected bygenerating a standard curve of fluorescence versus temperature for aprobe comprising a pair of interactive labels in a FRET or fluorescencequenching assay, as described above (see FIG. 12e). A probe thatexhibits a change in fluorescence that correlates with a change intemperature (see FIG. 12e) (e.g., fluorescence increases as thetemperature of the FRET reaction is increased) is capable of forming asecondary structure.

[0441] VI. Measuring a Change in Secondary Structure

[0442] A “change” in secondary structure according to the invention isdetected by analyzing a probe comprising a pair of interactive labels ina FRET or fluorescence quenching assay at a particular temperature belowthe Tm of the probe, (e.g., the cleaving temperature), as describedabove, in the presence or absence of 100 nM to 10 μM of a target nucleicacid (typically the target nucleic acid is in a 2-4 molar excess overthe probe concentration, i.e., 250-500 nM target nucleic acid is used).

[0443] Alternatively, a change in the secondary structure of the probeis determined by modifying the method of Gelfand et al. (1999, Proc.Natl. Acad. Sci. USA, 96:6113), incorporated herein by reference, todetermine the fluorescence of a probe labeled with a pair of interactivelabels in the presence or absence of a target nucleic acid as describedhereinabove.

[0444] A “change” in secondary structure that occurs when a probeaccording to the invention binds to a target nucleic acid, is measuredas an increase in fluorescence, such that the level of fluorescenceafter binding of the probe to the target nucleic acid at the temperaturebelow the Tm of the probe, is greater than (e.g., at least 5%,preferably 5-20% and more preferably 25 or more) the level offluorescence observed in the absence of a target nucleic acid (see FIG.12g).

[0445] VII. Methods of Use

[0446] The invention provides for a method of generating a signalindicative of the presence of a target nucleic acid in a samplecomprising the steps of forming a labeled cleavage structure byincubating a target nucleic acid with a probe having a secondarystructure, as defined herein, that changes upon binding to a targetnucleic acid and comprising a binding moiety, and cleaving the cleavagestructure with a nuclease (e.g. a FEN nuclease). The method of theinvention can be used in a PCR based assay as described below.

[0447] A labeled cleavage structure comprising an upstreamoligonucleotide primer (for example A, FIG. 4), a 5′ end labeleddownstream oligonucleotide probe having a secondary structure thatchanges upon binding to a target nucleic acid and comprising a bindingmoiety (for example C in FIG. 4) and a target nucleic acid (for exampleB in FIG. 4) is formed as described above in the section entitled“Cleavage Structure”. Briefly, a cleavage structure is formed andcleaved in the presence of a target nucleic acid, in the presence orabsence of an upstream primer (for example A, FIG. 4), a labeleddownstream probe as defined herein (for example C, FIG. 4) amplificationprimers specific for the target nucleic acid, a nucleic acid polymerasedeficient in 5′ to 3′ exonuclease activity a nuclease (e.g. a FENnuclease) and an appropriate buffer (for example 10×Pfu buffer,Stratagene, Catalog #200536) in a PCR reaction with the followingthermocycling parameters: 95° C. for 2 minutes and 40 cycles of 95° C.for 15 sec (denaturation step), 60° C. for 60 sec (annealing step) and72° C. for 15 sec (extension step). During this reaction an upstreamoligonucleotide (for example A, FIG. 4) is extended such thatoligonucleotide A partially displaces the 5′ labeled end of a downstreamoligonucleotide probe according to the invention (for exampleoligonucleotide C, FIG. 4) and the resulting labeled structure iscleaved with a nuclease (e.g., a FEN nuclease) according to theinvention. Alternatively, a downstream probe comprising a secondarystructure, as defined herein, (including a stem loop, a hairpin, aninternal loop, a bulge loop, a branched structure and a pseudoknot) ormultiple secondary structures, cloverleaf structures, or any threedimensional structure, as defined herein, can be used. Bi-molecular ormultimolecular probes, as defined herein, can also be used. The releasedlabeled fragment is captured by specific binding of the binding moietyto a capture element on a solid support according to methods well knownin the art (see Sambrook et al., supra and Ausubel et al., supra).Alternatively, a cleavage structure can be formed and cleaved in thepresence of a nucleic acid polymerase that possesses 5′ to 3′exonuclease activity.

[0448] The methods of the invention can also be used in non-PCR basedapplications to detect a target nucleic acid, where such target may beimmobilized on a solid support. Methods of immobilizing a nucleic acidsequence on a solid support are known in the art and are described inAusubel F M et al. Current Protocols in Molecular Biology, John Wileyand Sons, Inc. and in protocols provided by the manufacturers, e.g. formembranes: Pall Corporation, Schleicher & Schuell, for magnetic beads:Dynal, for culture plates: Costar, Nalgenunc, and for other supportsuseful according to the invention, CPG, Inc. A solid support usefulaccording to the invention includes but is not limited to silica basedmatrices, membrane based matrices and beads comprising surfacesincluding, but not limited to any of the solid supports described abovein the section entitled, “Cleavage Structure” and including styrene,latex or silica based materials and other polymers. Magnetic beads arealso useful according to the invention. Solid supports can be obtainedfrom the above manufacturers and other known manufacturers.

[0449] The invention also provides for a non-PCR based assay fordetecting a target nucleic acid in solution. The method of the inventioncan be used to detect naturally occurring target nucleic acids insolution including but not limited to RNA and DNA that is isolated andpurified from cells, tissues, single cell organisms, bacteria orviruses. The method of the invention can also be used to detectsynthetic targets in solution, including but not limited to RNA or DNAoligonucleotides, and peptide nucleic acids (PNAs). Non-PCR assaysinclude but are not limited to detection assays involving isothermallinear or exponential amplification, where the amount of nucleic acidsynthesized by the 3′ -5′ synthetic activity increases linearly orexponentially, and a nuclease (e.g. a FEN nuclease) is used to cleavethe displaced strand during synthesis. One such example utilizes rollingcircle amplification.

[0450] In one embodiment of the invention, detection of a nucleic acidtarget sequence that is either immobilized or in solution can beperformed by incubating an immobilized nucleic acid target sequence or atarget nucleic acid in solution with an upstream oligonucleotide primerthat is complementary to the target nucleic acid (for example A, FIG. 4)and a downstream oligonucleotide probe having a secondary structure thatchanges upon binding to a target nucleic acid and comprising a bindingmoiety, that is complementary to the target nucleic acid (for example C,FIG. 4), a nuclease (e.g. a FEN nuclease) and a nucleic acid polymerasethat possesses or lacks 5′ to 3′ exonuclease activity. The downstreamprobe is either end labeled at the 5′ or 3′ end, or is labeledinternally. Alternatively, a downstream probe comprising a secondarystructure, as defined herein, (including a stem loop, a hairpin, aninternal loop, a bulge loop, a branched structure and a pseudoknot) ormultiple secondary structures, cloverleaf structures, or any threedimensional structure, as defined herein, can be used. Bi-molecular ormultimolecular probes, as defined herein, can also be used. Detection ofa released labeled fragment that is captured by binding of the bindingmoiety to a capture element involves isotopic, enzymatic, orcolorimetric methods appropriate for the specific label that has beenincorporated into the probe and well known in the art (for example,Sambrook et al., supra, Ausubel et al., supra). Labels useful accordingto the invention and methods for the detection of labels usefulaccording to the invention are described in the section entitled“Cleavage Structure”. Alternatively, the downstream probe furthercomprises a pair of interactive signal generating labeled moieties (forexample a dye and a quencher) that are positioned such that when theprobe is intact, the generation of a detectable signal is quenched, andwherein the pair of interactive signal generating moieties are separatedby a nuclease cleavage site (e.g. a FEN nuclease cleavage site). Inanother embodiment, the downstream probe further comprises a pair ofinteractive signal generating labeled moieties (for example a dye and aquencher) that are positioned such that when the probe is not hybridizedto the target nucleic acid, the generation of a detectable signal isquenched. Upon cleavage by a nuclease (e.g. a FEN nuclease), the twosignal generating moieties are separated from each other and adetectable signal is produced. The presence of a pair of interactivesignal generating labeled moieties, as described above, allows fordiscrimination between annealed, uncleaved probe that may bind to acapture element, and released labeled fragment that is bound to acapture element. Nucleic acid polymerases that are useful for detectingan immobilized nucleic acid target sequence or a nucleic acid targetsequence in solution according to the method of the invention includemesophilic, thermophilic or hyper-thermophilic DNA polymerases lacking5′ to 3′ exonucleolytic activity (described in the section entitled,“Nucleic Acid Polymerases)”. Any nucleic acid polymerase that possess 5′to 3′ exonuclease activity is also useful according to the invention.

[0451] According to this non-PCR based method, the amount of a targetnucleic acid that can be detected is preferably about 1 pg to 1 μg, morepreferably about 1 pg to 10 ng and most preferably about 1 pg to 10 pg.Alternatively, this non-PCR based method can measure or detectpreferably about 1 molecule to 10²⁰ molecules, more preferably about 100molecules to 10¹⁷ molecules and most preferably about 1000 molecules to10¹⁴ molecules.

[0452] The invention also provides for a method of detecting a targetnucleic acid in a sample wherein a cleavage structure is formed asdescribed in the section entitled, “Cleavage Structure”, and the targetnucleic acid is amplified by a non-PCR based method including but notlimited to an isothermal method, for example rolling circle,Self-sustained Sequence Replication Amplification (3SR), Transcriptionbased amplification system (TAS), and Strand Displacement Amplification(SDA) and a non-isothermal method, for example Ligation chain reaction(LCR). A nuclease (e.g., a FEN nuclease) useful for non-PCRamplification methods will be active at a temperature range that isappropriate for the particular amplification method that is employed.

[0453] In the amplification protocols described below, samples whichneed to be prepared in order to quantify the target include: samples,no-template controls, and reactions for preparation of a standard curve(containing dilutions over the range of six orders of magnitude of asolution with a defined quantity of target).

[0454] Strand Displacement Amplification (SDA) is based on the abilityof a restriction enzyme to nick the unmodified strand of ahemiphosphorothioate form of its recognition site. The appropriate DNApolymerase will initiate replication at this nick and displace thedownstream non-template strand (Walker, 1992, Proc. Natl. Acad. Sci.USA, 89: 392, and PCR Methods and Applications 3: 1-6, 1993). Thepolymerases (Bca and Bst) which are used according to the method of SDAcan also be used in nuclease (e.g. FEN nuclease) directed cleavageaccording to the invention. According to the method of the invention, amolecular beacon is replaced by a nuclease (e.g., a FEN nuclease) activeat 42° C. and a cleavable probe having a secondary structure thatchanges upon binding to a target nucleic acid and further comprising abinding moiety, and comprising a cleavage structure according to theinvention.

[0455] A molecular beacon (Mb) is a fluorogenic probe which forms astem-loop structure is solution. Typically: 5′-fluorescent dye (e.g.FAM), attached to the 5′-stem region (5-7 nt), the loop region(complementary to the target, 20 to 30 nt), the 3′-stem region(complementary to the 5′-stem region), and the quencher (e.g. DABCYL).If no target is present, the MB forms its stem, which brings dye andquencher into close proximity, and therefore no fluorescence is emitted.When an MB binds to its target, the stem is opened, dye is spatiallyseparated from the quencher, and therefore the probe emits fluorescence(Tyagi S and Kramer F R, Nature Biotechnology 14: 303-308 (1996) andU.S. Pat. No. 5,925,517).

[0456] Strand Displacement Amplification (SDA) is essentially performedas described by Spargo et al., Molecular and Cellular Probes 10: 247-256(1996). The enzymes used include restriction endonuclease BsoBI (NewEngland Biolabs), DNA polymerase 5′-exo- Bca (PanVera Corporation). Thetarget is an insertion-like element (IS6110) found in the Mycobacteriumtuberculosis (Mtb) genome. The primers used are B1: cgatcgagcaagcca, B2:cgagccgctcgctg, S1: accgcatcgaatgcatgtctcgggtaaggcgtactcgacc and S2:cgattccgctccagacttctcgggtgtactgagatcccct. The Mycobacterium tuberculosisgenomic DNA is serially diluted in human placental DNA. SDA is performedin 50 μl samples containing 0 to 1000 Mtb genome equivalents, 500 nghuman placental DNA, 160 units BsoB1, 8 units of 5′-exo- Bca, 1.4 mMeach dCTPalphaS, TTP, dGTP, dATP, 35 mM K₂PO₄, pH 7.6 0.1 mg/mlacetylated bovine serum albumin (BSA), 3 mM Tris-HCl, 10 mM MgCl₂, 11 mMNaCl, 0.3 mM DTT, 4 mM KCl, 4% glycerol, 0.008 mM EDTA, 500 nM primersS1 and S2 and 50 nM primers B1 and B2 (KCl, glycerol and EDTA arecontributed by the BsoB1 storage solution). The samples (35 μl) wereheated in a boiling water bath for 3 minutes before the addition ofBsoB1 and 5′-exo Bca (10.7 units/μl BsoB1 and 0.53 units/μl 5′-exo Bcain 15 μl of New England Biolabs Buffer 2 (20 mM Tris-HCl pH 7.9, 10 mMMgCl₂, 50 mM NaCl, 1 mM DTT). Incubation is at 60° C. for 15 minutes,followed by 5 minutes in a boiling water bath.

[0457] Five μl of each sample in duplicate are removed for detection.Each reaction contains 1×Cloned Pfu buffer, 3.0 mM MgCl₂, 200 μM of eachdNTP, 5 units exo- Pfu, 23 ng Pfu FEN-1, 1 ng PEF, 300 nM each upstreamprimer: aaggcgtactcgacctgaaa and fluorogenic probe (for exampleFAM-DABCYL): accatacggataggggatctc. The reactions are subjected to onecycle in a thermal cycler: 2 minutes at 95° C., 1 minute at 55° C., 1minute at 72° C. The fluorescence is then determined in a fluorescenceplate reader, such as Stratagene's FluorTracker or PE Biosystems' 7700Sequence Detection System in Plate-Read Mode. The method of theinvention can also be performed with a polymerase that exhibits 5′ to 3′exonuclease activity and any nuclease included in the section entitled,“Nucleases”.

[0458] According to the method of nucleic acid sequence-basedamplification (NASBA), molecular beacons are used for quantification ofthe NASBA RNA amplicon in real-time analysis (Leone, et al., 1998,Nucleic Acids Res. 26: 2150). According to the method of the invention,NASBA can be carried out wherein the molecular beacon probe is replacedby a nuclease (e.g. a FEN nuclease) cleavable probe having a secondarystructure that changes upon binding to a target nucleic acid andcomprising a binding moiety, and further comprising a cleavage structureaccording to the invention and a nuclease (e.g. a FEN nuclease) activeat 41° C.

[0459] NASBA amplification is performed essentially as described byLeone G, et al., Nucleic Acids Res. 26: 2150-2155 (1998). Genomic RNAfrom the potato leafroll virus (PLRV) is amplified using the PD415 orPD416 (antisense) and the PD417 (sense) primers, which are described indetail in Leone G et al., J. Virol. Methods 66: 19-27 (1997). Each NASBAreaction contains a premix of 6 μl of sterile water, 4 μl of 5×NASBAbuffer (5×NASBA buffer is 200 mM Tris-HCl, pH 8.5, 60 mM MgCl₂, 350 mMKCl, 2.5 mM DTT, 5 mM each of dNTP, 10 mM each of ATP, UTP and CTP, 7.5mM GTP and 2.5 mM ITP), 4 μl of 5×primer mix (75% DMSO and 1 μM each ofantisense and sense primers). The premix is divided into 14 μl aliquots,to which 1 μl of PLRV target is added. After incubation for 5 minutes at65° C. and cooling to 41° C. for 5 minutes, 5 μl of enzyme mix is added(per reaction 375 mM sorbitol, 2.1 μg BSA, 0.08 units of RNase H(Pharmacia), 32 units of T7 RNA polymerase (Pharmacia) and 6.4 units ofAMV-RT (Seigakaku)). Amplification is for 90 minutes at 41° C.

[0460] Five μl of each sample in duplicate are removed for detection.Each reaction contains 1×Cloned Pfu buffer, 3.0 mM MgCl₂, 200 μM of eachdNTP, 5 units exo- Pfu, 23 ng Pfu FEN-1, 1 ng PEF, 300 nM each upstreamprimer PD415 or PD416 and the fluorogenic probe (for exampleFAM-DABCYL): gcaaagtatcatccctccag. The reactions are subjected to onecycle in a thermal cycler: 2 minutes at 95° C., 1 minute at 55° C., 1minute at 72° C. The fluorescence in then determined in a fluorescenceplate reader, such as Stratagene's FluorTracker or PE Biosystems' 7700Sequence Detection System in Plate-Read Mode.

[0461] Generally, according to these methods wherein amplificationoccurs by a non-PCR based method, amplification may be carried out inthe presence of a nuclease (e.g. a FEN nuclease), and amplification andcleavage by the nuclease (e.g. a FEN nuclease) occur simultaneously.Detection of released labeled fragments captured by binding of a bindingmoiety to a capture element on a solid support is performed as describedin the section entitled “Cleavage Structure” and may occur concurrentlywith (real time) or after (end-point) the amplification and cleavageprocess have been completed.

[0462] Endpoint assays can be used to quantify amplified target producedby non-PCR based methods wherein the amplification step is carried outin the presence of a nuclease (e.g., a FEN nuclease) (described above).

[0463] Endpoint assays include, but are not limited to the following.

[0464] A. Ligation chain reaction (LCR), as described in Landegren, etal., 1988, Science, 241: 1077 and Barany, PCR Methods and Applications1: 5-16 (1991). An LCR product useful according to the invention will belong enough such that the upstream primer and the labeled downstreamprobe are separated by a gap larger than 8 nucleotides to allow forefficient cleavage by a nuclease (e.g. a FEN nuclease).

[0465] B. Self-sustained sequence replication amplification (3SR) Fahy,et al. PCR Methods and Applications 1: 25-33 (1991). Self-SustainedSequence Replication Amplification (3SR) is a technique which is similarto NASBA. Ehricht R, et al., Nucleic Acids Res. 25: 4697-4699 (1997)have evolved the 3SR procedure to a cooperatively coupled in vitroamplification system (CATCH). Thus, in one embodiment of the invention,a molecular beacon probe is used for real-time analysis of an RNAamplicon by CATCH. The synthetic target amplified has the sequence:cctctgcagactactattacataatacgactcactatagggatctgcacgtattagcctatagtgagthgtattaataggaaacaccaaagatgatatttcgtcacagcaagaatteagg.The 3SR reactions contain 40 mM Tris-HCl pH 8.0, 5 mM KCl, 30 mM MgCl₂,1 mM of each dNTP, 1 nM of the double stranded target, 2 μM P1:cctctgcagactactattac and P2: cctgaattcttgctgtgacg, 5 mM DTT, 2 mMspermidine, 6 units/ul His tagged HIV-1 reverse transcriptase, 3units/ul T7-RNA polymerase and 0.16 units/ul Escherichia coli RNase H.The 100 ul reactions are incubated for 30 minutes at 42° C.

[0466] Five μl of each sample in duplicate are removed for detection.Each reaction contains 1×Cloned Pfu buffer, 3.0 mM MgCl₂, 200 μM of eachdNTP, 5 units exo- Pfu, 23 ng Pfu FEN-1, 1 ng PEF, 300 nM each upstreamprimer P1 and fluorogenic probe (for example FAM-DABCYL):taggaaacaccaaagatgatattt. The reactions are subjected to one cycle in athermal cycler: 2 minutes at 95° C., 1 minute at 55° C., 1 minute at 72°C. The fluorescence in then determined in a fluorescence plate reader,such as Stratagene's FluorTracker or PE Biosystems' 7700 SequenceDetection System in Plate-Read Mode. The method of 3SR can also becarried out with a polymerase that exhibits 5′ to 3′ exonucleaseactivity and any nuclease described in the section entitled,“Nucleases”.

[0467] C. Rolling circle amplification is described in U.S. Pat. No.5,854,033 and the related Ramification-Extension Amplification Method(RAM) (U.S. Pat. No. 5,942,391). Rolling circle amplification adapted tothe invention is described below.

[0468] Real-time assays can also be used to quantify amplified targetproduced by non-PCR based methods wherein the amplification step iscarried out in the presence of a nuclease (e.g. a FEN nuclease)(described above). The method of rolling circle amplification (U.S. Pat.No. 5,854,033) is adapted to include secondary primers for amplificationand detection, in conjunction with a nuclease (e.g. a FEN nuclease) anda cleavable probe having a secondary structure that changes upon bindingto a target nucleic acid and comprising a binding moiety, and furthercomprising a cleavage structure according to the invention, and iscarried out at temperatures between 50-60° C. The cleavage pattern of anuclease (e.g. a FEN nuclease) can be altered by the presence of asingle mismatched base located anywhere between 1 and 15 nucleotidesfrom the 5′ end of the primer wherein the DNA primer is otherwise fullyannealed. Typically, on a fully annealed substrate, a nuclease (e.g. aFEN nuclease) will exonucleolytically cleave the 5′ most nucleotide.However, a single nucleotide mismatch up to 15 nucleotides in from the5′ end promotes endonucleolytic cleavages. This constitutes a 5′proofreading process in which the mismatch promotes the nuclease actionthat leads to its removal. Thus, the mechanism of nuclease (e.g. FENnuclease) cleavage is shifted from predominantly exonucleolytic cleavageto predominantly endonucleolytic cleavage simply by the presence of asingle mismatched base pair. Presumably this occurs because a mismatchallows a short flap to be created (Rumbaugh et al., 1999, J. Biol.Chem., 274:14602).

[0469] The method of the invention can be used to generate a signalindicative of the presence of a sequence variation in a target nucleicacid, wherein a labeled cleavage structure comprising a fully annealedDNA primer is formed by incubating a target nucleic acid with a probehaving a secondary structure that changes upon binding to a targetnucleic acid and comprising a binding moiety (as described in thesection entitled, “Cleavage Structure”) and cleaving the labeledcleavage structure with a nuclease (e.g. a FEN nuclease) wherein therelease of labeled fragments comprising endonucleolytic cleavageproducts, and the detection of released fragments that are captured bybinding of a binding moiety to a capture element on a solid support, isindicative of the presence of a sequence variation. Released labeledfragments are detected as described in the section entitled, “CleavageStructure”.

[0470] V. Samples

[0471] The invention provides for a method of detecting or measuring atarget nucleic acid in a sample, as defined herein. As used herein,“sample” refers to any substance containing or presumed to contain anucleic acid of interest (a target nucleic acid) or which is itself atarget nucleic acid, containing or presumed to contain a target nucleicacid of interest. The term “sample” thus includes a sample of targetnucleic acid (genomic DNA, cDNA or RNA), cell, organism, tissue, fluidor substance including but not limited to, for example, plasma, serum,spinal fluid, lymph fluid, synovial fluid, urine, tears, stool, externalsecretions of the skin, respiratory, intestinal and genitourinarytracts, saliva, blood cells, tumors, organs, tissue, samples of in vitrocell culture constituents, natural isolates (such as drinking water,seawater, solid materials,) microbial specimens, and objects orspecimens that have been “marked” with nucleic acid tracer molecules.

EXAMPLES

[0472] The invention is illustrated by the following nonlimitingexamples wherein the following materials and methods are employed. Theentire disclosure of each of the literature references cited hereinafterare incorporated by reference herein.

Example 1

[0473] Probe Design and Preparation

[0474] The invention provides for a probe having a secondary structurethat changes upon binding of the probe to a target nucleic acid andcomprising a binding moiety.

[0475] A probe according to one embodiment of the invention is 5-250nucleotides in length and ideally 17-40 nucleotides in length, and has atarget nucleic acid binding sequence that is from 7 to about 140nucleotides, and preferably from 10 to about 140 nucleotides. Probes mayalso comprise non-covalently bound or covalently bound subunits.

[0476] One embodiment of a probe comprises a first complementary nucleicacid sequence (for example, b in FIG. 4) and a second complementarynucleic acid sequence (for example, b′ in FIG. 4). In one embodimentwherein the probe is unimolecular, the first and second complementarynucleic acid sequences are in the same molecule. In one embodiment, theprobe is labeled with a fluorophore and a quencher (for example,tetramethylrhodamine and DABCYL, or any of the fluorophore and quenchermolecules described herein (see the section entitled “How To Prepare aLabeled Cleavage Structure”). A probe according to the invention islabeled with an appropriate pair of interactive labels (e.g., a FRETpair or a non-FRET pair). The location of the interactive labels on theprobe is such that an appropriate spacing of the labels on the probe ismaintained to permit the separation of the labels when the probeundergoes a change in the secondary structure of the probe upon bindingto a target nucleic acid. For example, the donor and quencher moietiesare positioned on the probe to quench the generation of a detectablesignal when the probe is not bound to the target nucleic acid.

[0477] The probe further comprises a binding moiety (for example ab inFIG. 4, comprising a nucleic acid sequence, i.e.,5′AGCTACTGATGCAGTCACGT3′). In one embodiment of the invention, uponhybridization to a target nucleic acid, the probe according to theinvention, forms a cleavage structure comprising a 5′ flap (e.g., ab inFIG. 4). The flap of the cleavage structure thus comprises the bindingmoiety of the probe. Cleaving is performed at a cleaving temperature,and the secondary structure of the probe when not bound to the targetnucleic acid is stable at or below the cleaving temperature. Uponcleavage of the hybridized probe by a nuclease, the binding moiety isreleased and binds specifically to a capture element comprising anucleic acid sequence, i.e., 5′TCGATGACTACGTCAGTGCA3′. According to thisembodiment, the binding moiety comprises two regions (for example a andb in FIG. 4). The region of a “binding moiety” that is not a“complementary nucleic acid sequence”, as defined herein, (e.g., a inFIG. 4), is from 1-60 nucleotides, preferably from 1-25 nucleotides andmost preferably from 1-10 nucleotides in length. Region b is one of atleast two complementary nucleic acid sequences of the probe, as definedherein, the length of which is described in detail below.

[0478] In one embodiment, in the absence of the target nucleic acid theprobe folds back on itself to generate an antiparallel duplex structurewherein the first and second complementary nucleic acid sequences annealby the formation of hydrogen bonds to form a secondary structure. Thesecondary structure of the probe is detected by performing a FRET orfluorescence quenching assay at different temperatures, includingtemperatures that are above and below the Tm of the probe, as describedherein. A probe that exhibits a change in fluorescence that correlateswith a change in temperature (e.g., fluorescence increases as thetemperature of the FRET reaction is increased), greater than a change influorescence simply due to thermal effects on the efficiency offluorophore emission, has secondary structure. Secondary structure iseliminated at a temperature wherein the maximal level of fluorescence isdetected (e.g., fluorescence does not increase above this level atincreased temperatures). The stability of the secondary structure of theprobe is determined in a melting temperature assay or by FRET orfluorescence quenching assay, as described herein.

[0479] As a result of the change in the secondary structure of theprobe, the binding moiety becomes accessible for cleavage by a nuclease.In the presence of the target nucleic acid, and at a temperature that isselected according to the factors that influence the efficiency andselectivity of hybridization of the probe to the target nucleic acid,(e.g., primer length, nucleotide sequence and/or composition, buffercomposition, as described in the section entitled, “Primers and ProbesUseful According to the Invention”) to permit specific binding of theprobe and the target nucleic acid, the probe binds to the target nucleicacid and undergoes a change in the secondary structure. A change in thesecondary structure of the probe can be determined by FRET orfluorescence quenching, as described herein.

[0480] In one embodiment, first and second complementary nucleic acidsequences are 3-25, preferably 4-15 and more preferably 5-11 nucleotideslong. The length of the first and second complementary nucleic acidsequences is selected such that the secondary structure of the probewhen not bound to the target nucleic acid is stable at the temperatureat which cleavage of a cleavage structure comprising the probe bound toa target nucleic acid is performed. As the target nucleic acid bindingsequence increases in size up to 100 nucleotides, the length of thecomplementary nucleic acid sequences may increase up to 15-25nucleotides. For a target nucleic acid binding sequence greater than 100nucleotides, the length of the complementary nucleic acid sequences arenot increased further.

[0481] Alternatively, an allele discriminating probe having secondarystructure and comprising a binding moiety is prepared.

[0482] In one embodiment, an allele discriminating probe according tothe invention preferably comprises a target nucleic acid bindingsequence from 6 to 50 and preferably from 7 to 25 nucleotides, andsequences of the complementary nucleic acid sequences from 3 to 8nucleotides. The guanosine-cytidine content of the secondary structureand probe-target hybrids, salt, and assay temperature are considered,for example magnesium salts have a strong stabilizing effect, whendesigning short, allele-discriminating probes.

[0483] An allele-discriminating probe with a target nucleic acid bindingsequence near the upper limits of 50 nucleotides long, is designed suchthat the single nucleotide mismatch to be discriminated against occursat or near the middle of the target nucleic acid binding sequence. Forexample, probes comprising a sequence that is 21 nucleotides long arepreferably designed so that the mismatch occurs opposite one of the 14most centrally located nucleotides of the target nucleic acid bindingsequence and most preferably opposite one of the 7 most centrallylocated nucleotides.

Example 2

[0484] Probe Design and Preparation

[0485] The invention provides for a probe having a secondary structurethat changes upon binding of the probe to a target nucleic acid andcomprising a binding moiety.

[0486] A probe according to one embodiment of the invention is 5-250nucleotides in length and ideally 17-40 nucleotides in length, and has atarget nucleic acid binding sequence that is from 7 to about 140nucleotides, and preferably from 10 to about 140 nucleotides. Probes mayalso comprise non-covalently bound or covalently bound subunits.

[0487] One embodiment of a probe comprises a first complementary nucleicacid sequence (for example, b in FIG. 4) and a second complementarynucleic acid sequence (for example, b′ in FIG. 4). In one embodimentwherein the probe is unimolecular, the first and second complementarynucleic acid sequences are in the same molecule. In one embodiment, theprobe is labeled with a fluorophore and a quencher (for example,tetramethylrhodamine and DABCYL, or any of the fluorophore and quenchermolecules described herein (see the section entitled “How To Prepare aLabeled Cleavage Structure”). A probe according to the invention islabeled with an appropriate pair of interactive labels (e.g., a FRETpair or a non-FRET pair). The location of the interactive labels on theprobe is such that an appropriate spacing of the labels on the probe ismaintained to permit the separation of the labels when the probeundergoes a change in the secondary structure of the probe upon bindingto a target nucleic acid. For example, the donor and quencher moietiesare positioned on the probe to quench the generation of a detectablesignal when the probe is not bound to the target nucleic acid.

[0488] The probe further comprises a tag comprising the lac repressorprotein. In one embodiment of the invention, upon hybridization to atarget nucleic acid, the probe forms a cleavage structure comprising a5′ flap (e.g., ab in FIG. 4). Cleaving is performed at a cleavingtemperature, and the secondary structure of the probe when not bound tothe target nucleic acid is stable at or below the cleaving temperature.Upon cleavage of the hybridized probe by a nuclease, the lac repressorprotein binds specifically to a capture element comprising the doublestranded DNA sequence recognized and bound specifically by the lacrepressor protein:

[0489] AATTGTGAGCGGATAACAATT

[0490] TTAACACTCGCCTATTGTTAA.

[0491] In one embodiment, in the absence of the target nucleic acid theprobe folds back on itself to generate an antiparallel duplex structurewherein the first and second complementary nucleic acid sequences annealby the formation of hydrogen bonds to form a secondary structure. Thesecondary structure of the probe is detected by performing a FRET orfluorescence quenching assay at different temperatures, includingtemperatures that are above and below the Tm of the probe, as describedherein. A probe that exhibits a change in fluorescence that correlateswith a change in temperature (e.g., fluorescence increases as thetemperature of the FRET reaction is increased), greater than a change influorescence simply due to thermal effects on the efficiency offluorophore emission, has secondary structure. Secondary structure iseliminated at a temperature wherein the maximal level of fluorescence isdetected (e.g., fluorescence does not increase above this level atincreased temperatures). The stability of the secondary structure of theprobe is determined in a melting temperature assay or by FRET orfluorescence quenching assay, as described herein.

[0492] As a result of the change in the secondary structure of theprobe, the tag becomes accessible for cleavage by a nuclease. In thepresence of the target nucleic acid, and at a temperature that isselected according to the factors that influence the efficiency andselectivity of hybridization of the probe to the target nucleic acid,(e.g., primer length, nucleotide sequence and/or composition, buffercomposition, as described in the section entitled, “Primers and ProbesUseful According to the Invention”) to permit specific binding of theprobe and the target nucleic acid, the probe binds to the target nucleicacid and undergoes a change in the secondary structure. A change in thesecondary structure of the probe can be determined by FRET orfluorescence quenching, as described herein.

[0493] In one embodiment, first and second complementary nucleic acidsequences are 3-25, preferably 4-15 and more preferably 5-11 nucleotideslong. The length of the first and second complementary nucleic acidsequences is selected such that the secondary structure of the probewhen not bound to the target nucleic acid is stable at the temperatureat which cleavage of a cleavage structure comprising the probe bound toa target nucleic acid is performed. As the target nucleic acid bindingsequence increases in size up to 100 nucleotides, the length of thecomplementary nucleic acid sequences may increase up to 15-25nucleotides. For a target nucleic acid binding sequence greater than 100nucleotides, the length of the complementary nucleic acid sequences arenot increased further.

[0494] Alternatively, an allele discriminating probe having secondarystructure and comprising a binding moiety is prepared.

[0495] In one embodiment, an allele discriminating probe according tothe invention preferably comprises a target nucleic acid bindingsequence from 6 to 50 and preferably from 7 to 25 nucleotides, andsequences of the complementary nucleic acid sequences from 3 to 8nucleotides. The guanosine-cytidine content of the secondary structureand probe-target hybrids, salt, and assay temperature are considered,for example magnesium salts have a strong stabilizing effect, whendesigning short, allele-discriminating probes.

[0496] An allele-discriminating probe with a target nucleic acid bindingsequence near the upper limits of 50 nucleotides long, is designed suchthat the single nucleotide mismatch to be discriminated against occursat or near the middle of the target nucleic acid binding sequence. Forexample, probes comprising a sequence that is 21 nucleotides long arepreferably designed so that the mismatch occurs opposite one of the 14most centrally located nucleotides of the target nucleic acid bindingsequence and most preferably opposite one of the 7 most centrallylocated nucleotides.

Example 3

[0497] A target nucleic acid can be detected and/or measured by thefollowing method. A labeled cleavage structure is formed prior to theaddition of a FEN nuclease by heating at 95° C. for 5 minutes and thencooling to approximately 50-60° C. (a) a sample containing a targetnucleic acid (B in FIG. 4) with (b) an upstream oligonucleotide thatspecifically hybridizes to the target nucleic acid, (A, in FIG. 4), and(c) a downstream, 5′ end labeled oligonucleotide probe having asecondary structure that changes upon binding of the probe to the targetnucleic acid and comprising a binding moiety (for example ab in FIG. 4,comprising a nucleic acid sequence, i.e., 5′AGCTACTGATGCAGTCACGT3′),wherein the probe specifically hybridizes to a region of the targetnucleic acid that is downstream of the hybridizing region ofoligonucleotide A. A polymerase that lacks a 5′ to 3′ exonucleaseactivity but that possesses a 3′ to 5′ DNA synthetic activity, such asthe enzyme a)Yaq exo-, (prepared by mutagenesis using the StratageneQuikChange Site-Directed Mutagenesis kit, catalog number #200518, tomodify Taq polymerase (Tabor and Richardson, 1985, Proc. Natl. Acad.Sci. USA, 82:1074)), a mutant form of Taq polymerase that lacks 5′ to 3′exonuclease activity, b) Pfu, or c) a mutant form of Pfu polymerase thatlacks 3′ to 5′ exonuclease activity (exo- Pfu) is added and incubatedunder conditions that permit the polymerase to extend oligonucleotide Asuch that it partially displaces the 5′ end of oligonucleotide C (forexample 72° C. in 1×Pfu buffer (Stratagene) for 5 minutes to 1 hour. Thedisplaced region of oligonucleotide C forms a 5′ flap that is cleavedupon the addition of a FEN nuclease. Alternatively, extension isperformed with a polymerase that exhibits 5′ to 3′ exonuclease activityand with any nuclease included in the section entitled, “Nucleases”.

[0498] A mutant form of Taq polymerase that lacks a 5′ to 3′ exonucleaseactivity but that possesses a 3′ to 5′ DNA synthetic activity comprisesthe following mutation: D144S/F667Y Taq wherein D144S eliminates 5′ to3′ exonuclease activity and F667Y improves ddNTP incorporation.

[0499] Exo- mutants of PolI polymerase can be prepared according to themethod of Xu et al., 1997, J. Mol. Biol., 268: 284.

[0500] A labeled cleavage structure according to the invention iscleaved with a preparation of PfuFEN-1 (i.e. cloned Pyrococcus furiosusFEN-1 that is prepared as described below in Example 9). Cleaving isperformed at a cleaving temperature, and the secondary structure of theprobe when not bound to the target nucleic acid is stable at or belowthe cleaving temperature. Cleavage is carried out by adding 2 μl ofPfuFEN-1 to a 7 μl reaction mixture containing the following:

[0501] 3 μl cleavage structure (10 ng-10 μg)

[0502] 0.7 μl 10×FEN nuclease buffer (10×FEN nuclease buffer contains500 mM Tris-HCl pH 8.0, 100 mM MgCl₂)

[0503] 2.00 μl PfuFEN-1 enzyme or H₂O

[0504] 1.3 μl H₂O

[0505]7.00 μl total volume

[0506] Samples are incubated for one hour at 50° C. in a Robocyler 96hot top thermal cycler. Following the addition of 2 μl of SequencingStop dye solution (included in the Stratagene Cyclist DNA sequencingkit, catalog #200326), samples are heated at 99° C. for five minutes.Released, labeled, fragments comprising the binding moiety are bound viabinding of the binding moiety to a capture element comprising a nucleicacid sequence, i.e., 5′TCGATGACTACGTCAGTGCA3′, on a solid support. Inone embodiment, the labeled fragments are eluted from the captureelement by, for example, decreasing the salt concentration (stringenthybridization conditions typically include salt concentrations of lessthan about 1M, more usually less than about 500 mM and preferably lessthan about 200 mM) or by adding an excess of unlabeled, competitorfragment. Samples containing eluted labeled fragments are analyzed bygel electrophoresis as follows. Samples are loaded on an eleven inchlong, hand-poured, 20% acrylamide/bis acrylamide, 7M urea gel. The gelis run at 20 watts until the bromophenol blue has migrated approximately⅔ the total distance. The gel is removed from the glass plates andsoaked for 10 minutes in fix solution (15% methanol, 5% acetic acid) andthen for 10 minutes in water. The gel is placed on Whatmann 3 mm paper,covered with plastic wrap and dried for 2 hours in a heated vacuum geldryer (˜80° C.). The gel is exposed overnight to X-ray film to detectthe presence of a signal that is indicative of the presence of a targetnucleic acid.

[0507] Alternatively, extension is performed with a polymerase thatexhibits 5′ to 3′ exonuclease activity and with any nuclease included inthe section entitled, “Nucleases”.

Example 4

[0508] A target nucleic acid can be detected and/or measured by thefollowing method. A labeled cleavage structure is formed prior to theaddition of a FEN nuclease by heating at 95° C. for 5 minutes and thencooling to approximately 50-60° C. (a) a sample containing a targetnucleic acid (B in FIG. 4) with (b) an upstream oligonucleotide thatspecifically hybridizes to the target nucleic acid, (A, in FIG. 4), and(c) a downstream, 5′ end labeled oligonucleotide probe having asecondary structure that changes upon binding of the probe to the targetnucleic acid and comprising a lac repressor protein tag, wherein theprobe specifically hybridizes to a region of the target nucleic acidthat is downstream of the hybridizing region of oligonucleotide A. Apolymerase that lacks a 5′ to 3′ exonuclease activity but that possessesa 3′ to 5′ DNA synthetic activity, such as the enzyme a)Yaq exo-,(prepared by mutagenesis using the Stratagene QuikChange Site-DirectedMutagenesis kit, catalog number #200518, to modify Taq polymerase (Taborand Richardson, 1985, Proc. Natl. Acad. Sci. USA, 82:1074)), a mutantform of Taq polymerase that lacks 5′ to 3′ exonuclease activity, b) Pfu,or c) a mutant form of Pfu polymerase that lacks 3′ to 5′ exonucleaseactivity (exo- Pfu) is added and incubated under conditions that permitthe polymerase to extend oligonucleotide A such that it partiallydisplaces the 5′ end of oligonucleotide C (for example 72° C. in 1×Pfubuffer (Stratagene) for 5 minutes to 1 hour. The displaced region ofoligonucleotide C forms a 5′ flap that is cleaved upon the addition of aFEN nuclease. Alternatively, extension is performed with a polymerasethat exhibits 5′ to 3′ exonuclease activity and with any nucleaseincluded in the section entitled, “Nucleases”.

[0509] A mutant form of Taq polymerase that lacks a 5′ to 3′ exonucleaseactivity but that possesses a 3′ to 5′ DNA synthetic activity comprisesthe following mutation: D144S/F667Y Taq wherein D144S eliminates 5′ to3′ exonuclease activity and F667Y improves ddNTP incorporation.

[0510] Exo- mutants of Poll polymerase can be prepared according to themethod of Xu et al., 1997, J. Mol. Biol., 268: 284.

[0511] A labeled cleavage structure according to the invention iscleaved with a preparation of PfuFEN-1 (i.e. cloned Pyrococcus furiosusFEN-1 that is prepared as described below in Example 9). Cleaving isperformed at a cleaving temperature, and the secondary structure of theprobe when not bound to the target nucleic acid is stable at or belowthe cleaving temperature. Cleavage is carried out by adding 2 μl ofPfuFEN-1 to a 7 μl reaction mixture containing the following:

[0512] 3 μl cleavage structure (10 ng-10 μg)

[0513] 0.7 μl 10×FEN nuclease buffer (10×FEN nuclease buffer contains500 mM Tris-HCl pH 8.0, 100 mM MgCl₂)

[0514] 2.00 μl PfuFEN-1 enzyme or H₂O

[0515] 1.3 μl H₂O

[0516]7.00 μl total volume

[0517] Samples are incubated for one hour at 50° C. in a Robocyler 96hot top thermal cycler. Following the addition of 2 μl of SequencingStop dye solution (included in the Stratagene Cyclist DNA sequencingkit, catalog #200326), samples are heated at 99° C. for five minutes.Released, labeled, fragments comprising the lac repressor protein arebound via binding of the lac repressor protein to a capture elementcomprising the double stranded DNA sequence recognized by the lacrepressor protein:

[0518] AATTGTGAGCGGATAACAATT

[0519] ATTAACACTCGCCTATTGTTAA, on a solid support.

[0520] In one embodiment, the labeled fragments are eluted from thecapture element by, for example, altering the salt concentration, i.e.,decreasing the salt concentration (stringent hybridization conditionstypically include salt concentrations of less than about 1M, moreusually less than about 500 mM and preferably less than about 200 mM) orby adding an excess of competitor a)lac repressor protein or b) doublestranded DNA sequence recognized by the lac repressor protein. Samplescontaining eluted labeled fragments are analyzed by gel electrophoresisas follows. Samples are loaded on an eleven inch long, hand-poured, 20%acrylamide/bis acrylamide, 7M urea gel. The gel is run at 20 watts untilthe bromophenol blue has migrated approximately ⅔ the total distance.The gel is removed from the glass plates and soaked for 10 minutes infix solution (15% methanol, 5% acetic acid) and then for 10 minutes inwater. The gel is placed on Whatmann 3 mm paper, covered with plasticwrap and dried for 2 hours in a heated vacuum gel dryer (˜80° C.). Thegel is exposed overnight to X-ray film to detect the presence of asignal that is indicative of the presence of a target nucleic acid.

[0521] Alternatively, extension is performed with a polymerase thatexhibits 5′ to 3′ exonuclease activity and with any nuclease included inthe section entitled, “Nucleases”.

Example 5

[0522] A target nucleic acid can be detected and/or measured by thefollowing method. A labeled cleavage structure is formed prior to theaddition of a FEN nuclease by annealing at 95° C. for 5 minutes and thencooling to approximately 50-60° C. (a) a sample containing a targetnucleic acid (B in FIG. 4) with (b) an upstream oligonucleotide primerthat specifically hybridizes to the target nucleic acid, (A, in FIG. 4),and (c) a downstream, 5′ end labeled oligonucleotide probe having asecondary structure that changes upon binding of the probe to the targetnucleic acid and comprising a binding moiety (for example ab in FIG. 4,comprising a nucleic acid sequence 5′AGCTACTGATGCAGTCACGT3′), whereinthe probe specifically hybridizes to a region of the target nucleic acidthat is adjacent to the hybridizing region of oligonucleotide A andfurther comprises a 5′ region that does not hybridize to the targetnucleic acid and forms a 5′ flap. Annealing is carried out in thepresence of 1×Sentinal Molecular beacon core buffer or 10×Pfu buffer.

[0523] A labeled cleavage structure according to the invention iscleaved with a preparation of PfuFEN-1 (i.e. cloned Pyrococcus furiosusFEN-1 that is prepared as described below in Example 9). Cleaving isperformed at a cleaving temperature, and the secondary structure of theprobe when not bound to the target nucleic acid is stable at or belowthe cleaving temperature. Cleavage is carried out by adding 2 μl ofPfuFEN-1 to a 7 μl reaction mixture containing the following:

[0524] 3 μl cleavage structure (10 ng-10 μg)

[0525] 0.7 μl 10×FEN nuclease buffer (10×FEN nuclease buffer contains500 mM Tris-HCl pH 8.0, 100 mM MgCl₂)

[0526] 2.00 μl PfuFEN-1 enzyme or H₂O

[0527] 1.3 μl H₂O

[0528]7.00 μl total volume

[0529] Samples are incubated for one hour at 50° C. in a Robocyler 96hot top thermal cycler. Following the addition of 2 μl of SequencingStop dye solution (included in the Stratagene Cyclist DNA sequencingkit, catalog #200326), samples are heated at 99° C. for five minutes.Released, labeled, fragments comprising a binding moiety are bound viabinding of the binding moiety to a capture element comprising thesequence, 5′TCGATGACTACGTCAGTGCA3′, on a solid support. In oneembodiment, the labeled fragments are eluted from the capture elementby, for example, decreasing the salt concentration (stringenthybridization conditions typically include salt concentrations of lessthan about 1M, more usually less than about 500 mM and preferably lessthan about 200 mM) or by adding an excess of unlabeled, competitorfragment. Samples containing eluted labeled fragments are analyzed bygel electrophoresis as follows. Samples are loaded on an eleven inchlong, hand-poured, 20% acrylamide/bis acrylamide, 7M urea gel. The gelis run at 20 watts until the bromophenol blue has migrated approximately⅔ the total distance. The gel is removed from the glass plates andsoaked for 10 minutes in fix solution (15% methanol, 5% acetic acid) andthen for 10 minutes in water. The gel is placed on Whatmann 3 mm paper,covered with plastic wrap and dried for 2 hours in a heated vacuum geldryer (˜80° C.). The gel is exposed overnight to X-ray film to detectthe presence of a signal that is indicative of the presence of a targetnucleic acid.

[0530] Alternatively, extension is performed with a polymerase thatexhibits 5′ to 3′ exonuclease activity and with any nuclease included inthe section entitled, “Nucleases”.

Example 6

[0531] A target nucleic acid can be detected and/or measured by thefollowing method. A labeled cleavage structure is formed prior to theaddition of a FEN nuclease by annealing at 95° C. for 5 minutes and thencooling to approximately 50-60° C. (a) a sample containing a targetnucleic acid (B in FIG. 4) with (b) an upstream oligonucleotide primerthat specifically hybridizes to the target nucleic acid, (A, in FIG. 4),and (c) a downstream, 5′ end labeled oligonucleotide probe having asecondary structure that changes upon binding of the probe to the targetnucleic acid and comprising a lac repressor protein tag, wherein theprobe specifically hybridizes to a region of the target nucleic acidthat is adjacent to the hybridizing region of oligonucleotide A andfurther comprises a 5′ region that does not hybridize to the targetnucleic acid and forms a 5′ flap. Annealing is carried out in thepresence of 1×Sentinal Molecular beacon core buffer or 10×Pfu buffer.

[0532] A labeled cleavage structure according to the invention iscleaved with a preparation of PfuFEN-1 (i.e. cloned Pyrococcus furiosusFEN-1 that is prepared as described below in Example 9). Cleaving isperformed at a cleaving temperature, and the secondary structure of theprobe when not bound to the target nucleic acid is stable at or belowthe cleaving temperature. Cleavage is carried out by adding 2 μl ofPfuFEN-1 to a 7 μl reaction mixture containing the following:

[0533] 3 μl cleavage structure (10 ng-10 μg)

[0534] 0.7 μl 10×FEN nuclease buffer (10×FEN nuclease buffer contains500 mM Tris-HCl pH 8.0, 100 mM MgCl₂)

[0535] 2.00 μl PfuFEN-1 enzyme or H₂O

[0536] 1.3 μl H₂O

[0537]7.00 μl total volume

[0538] Samples are incubated for one hour at 50° C. in a Robocyler 96hot top thermal cycler. Following the addition of 2 μl of SequencingStop dye solution (included in the Stratagene Cyclist DNA sequencingkit, catalog #200326), samples are heated at 99° C. for five minutes.

[0539] Upon cleavage of the hybridized probe by a nuclease, the lacrepressor protein binds specifically to a capture element comprising thedouble stranded DNA sequence recognized by the lac repressor protein:

[0540] AATTGTGAGCGGATAACAATT

[0541] TTAACACTCGCCTATTGTTAA, on a solid support.

[0542] In one embodiment, the labeled fragments are eluted from thecapture element as described in Example 4, above. Samples containingeluted labeled fragments are analyzed by gel electrophoresis as follows.Samples are loaded on an eleven inch long, hand-poured, 20%acrylamide/bis acrylamide, 7M urea gel. The gel is run at 20 watts untilthe bromophenol blue has migrated approximately ⅔ the total distance.The gel is removed from the glass plates and soaked for 10 minutes infix solution (15% methanol, 5% acetic acid) and then for 10 minutes inwater. The gel is placed on Whatmann 3 mm paper, covered with plasticwrap and dried for 2 hours in a heated vacuum gel dryer (˜80° C.). Thegel is exposed overnight to X-ray film to detect the presence of asignal that is indicative of the presence of a target nucleic acid.

[0543] Alternatively, extension is performed with a polymerase thatexhibits 5′ to 3′ exonuclease activity and with any nuclease included inthe section entitled, “Nucleases”.

Example 7

[0544] A target nucleic acid can be detected and/or measured by thefollowing method. A labeled cleavage structure is formed prior to theaddition of a FEN nuclease by annealing at 95° C. for 5 minutes and thencooling to approximately 50-60° C. (a) a sample containing a targetnucleic acid (B in FIG. 4) with (b) a downstream, 5′ end labeledoligonucleotide probe having a secondary structure that changes uponbinding of the probe to the target nucleic acid and a binding moiety(for example ab in FIG. 4, comprising a nucleic acid sequence, i.e.,5′AGCTACTGATGCAGTCACGT3′), wherein the probe specifically hybridizes toa region of the target nucleic acid and comprises a 5′ region that doesnot hybridize to the target nucleic acid and forms a 5′ flap. Annealingis carried out in the presence of 1×Sentinal Molecular beacon corebuffer or 10×Pfu buffer.

[0545] A labeled cleavage structure according to the invention iscleaved with a nuclease that is capable of cleaving this cleavagestructure (e.g., Taq polymerase). Cleaving is performed at a cleavingtemperature, and the secondary structure of the probe when not bound tothe target nucleic acid is stable at or below the cleaving temperature.Cleavage is carried out by adding 2 μl of a nuclease to a 7 μl reactionmixture containing the following:

[0546] 3 μl cleavage structure (10 ng-10 μg)

[0547] 0.7 μl 10×nuclease buffer (500 mM Tris-HCl pH 8.0, 100 mM MgCl₂)

[0548] 2.00 μl nuclease or H₂O

[0549] 1.3 μl H₂O

[0550]7.00 μl total volume

[0551] Samples are incubated for one hour at 50° C. in a Robocyler 96hot top thermal cycler. Following the addition of 2 μl of SequencingStop dye solution (included in the Stratagene Cyclist DNA sequencingkit, catalog #200326), samples are heated at 99° C. for five minutes.Released, labeled, fragments comprising a binding moiety are bound viabinding of the binding moiety to a capture element comprising a nucleicacid sequence, i.e., 5′TCGATGACTACGTCAGTGCA3′, on a solid support. Inone embodiment, the labeled fragments are eluted from the captureelement by, for example, decreasing the salt concentration (stringenthybridization conditions typically include salt concentrations of lessthan about 1M, more usually less than about 500 mM and preferably lessthan about 200 mM) or by adding an excess of unlabeled, competitorfragment. Samples containing eluted labeled fragments are analyzed bygel electrophoresis as follows. Samples are loaded on an eleven inchlong, hand-poured, 20% acrylamide/bis acrylamide, 7M urea gel. The gelis run at 20 watts until the bromophenol blue has migrated approximately⅔ the total distance. The gel is removed from the glass plates andsoaked for 10 minutes in fix solution (15% methanol, 5% acetic acid) andthen for 10 minutes in water. The gel is placed on Whatmann 3 mm paper,covered with plastic wrap and dried for 2 hours in a heated vacuum geldryer (˜80° C.). The gel is exposed overnight to X-ray film to detectthe presence of a signal that is indicative of the presence of a targetnucleic acid.

[0552] Alternatively, extension is performed with a polymerase thatexhibits 5′ to 3′ exonuclease activity and with any nuclease included inthe section entitled, “Nucleases”.

Example 8

[0553] A target nucleic acid can be detected and/or measured by thefollowing method.

[0554] A labeled cleavage structure is formed prior to the addition of aFEN nuclease by annealing at 95° C. for 5 minutes and then cooling toapproximately 50-60° C. (a) a sample containing a target nucleic acid (Bin FIG. 4) with (b) a downstream, 5′ end labeled oligonucleotide probehaving a secondary structure that changes upon binding of the probe tothe target nucleic acid and a tag comprising the lac repressor protein,wherein the probe specifically hybridizes to a region of the targetnucleic acid and comprises a 5′ region that does not hybridize to thetarget nucleic acid and forms a 5′ flap. Annealing is carried out in thepresence of 1×Sentinal Molecular beacon core buffer or 10×Pfu buffer.

[0555] A labeled cleavage structure according to the invention iscleaved with a nuclease that is capable of cleaving this cleavagestructure (e.g., Taq polymerase). Cleaving is performed at a cleavingtemperature, and the secondary structure of the probe when not bound tothe target nucleic acid is stable at or below the cleaving temperature.Cleavage is carried out by adding 2 μl of a nuclease to a 7 μl reactionmixture containing the following:

[0556] 3 μl cleavage structure (10 ng-10 μg)

[0557] 0.7 μg 10×nuclease buffer (500 mM Tris-HCl pH 8.0, 100 mM MgCl₂)

[0558] 2.00 μl nuclease or H₂O

[0559] 1.3 μl H₂O

[0560]7.00 μl total volume

[0561] Samples are incubated for one hour at 50° C. in a Robocyler 96hot top thermal cycler. Following the addition of 2 μl of SequencingStop dye solution (included in the Stratagene Cyclist DNA sequencingkit, catalog #200326), samples are heated at 99° C. for five minutes.Upon cleavage of the hybridized probe by a nuclease, the lac repressorprotein binds specifically to a capture element comprising the doublestranded DNA sequence recognized by the lac repressor protein:

[0562] AATTGTGAGCGGATAACAATT

[0563] TTAACACTCGCCTATTGTTAA, on a solid support.

[0564] In one embodiment, the labeled fragments are eluted from thecapture element as described in Example 4, above. Samples containingeluted labeled fragments are analyzed by gel electrophoresis as follows.Samples are loaded on an eleven inch long, hand-poured, 20%acrylamide/bis acrylamide, 7M urea gel. The gel is run at 20 watts untilthe bromophenol blue has migrated approximately ⅔ the total distance.The gel is removed from the glass plates and soaked for 10 minutes infix solution (15% methanol, 5% acetic acid) and then for 10 minutes inwater. The gel is placed on Whatmann 3 mm paper, covered with plasticwrap and dried for 2 hours in a heated vacuum gel dryer (˜80° C.). Thegel is exposed overnight to X-ray film to detect the presence of asignal that is indicative of the presence of a target nucleic acid.

[0565] Alternatively, extension is performed with a polymerase thatexhibits 5′ to 3′ exonuclease activity and with any nuclease included inthe section entitled, “Nucleases”.

Example 9

[0566] Cloning Pfu FEN-1

[0567] A thermostable FEN nuclease enzyme useful according to theinvention can be prepared according to the following method.

[0568] The thermostable FEN nuclease gene can be isolated from genomicDNA derived from P. furiosus (ATCC#43587) according to methods of PCRcloning well known in the art. The cloned PfuFEN-1 can be overexpressedin bacterial cells according to methods well known in the art anddescribed below.

[0569] The following pCAL-n-EK cloning oligonucleotides were synthesizedand purified:

[0570] a. 5′GACGACGACAAGATGGGTGTCCCAATTGGTGAGATTATACCAAGAA AAG 3′ and

[0571] b. 5′GGAACAAGACCCGTTTATCTCTTGAACCAACTTTCAAGGGTTGATTG TTTTCCACT3′.

[0572] The Affinity® Protein Expression and Purification System wasobtained from Stratagene and used according to the manufacturer'sprotocols.

[0573] Amplification

[0574] The insert DNA was prepared by PCR amplification withgene-specific primers (oligonucleotides a and b, described above) thatinclude 12 and 13-nucleotide sequences at the 5′ ends that arecomplementary to the pCAL-n-EK vector single-stranded tails, thusallowing for directional cloning. The FEN-1 sequence was amplified fromgenomic DNA derived from P. furiosus by preparing amplificationreactions (five independent 100 μl reactions) containing:

[0575] 50 μl 10×cPfu Buffer (Stratagene)

[0576] 7.5 μl Pfu Genomic DNA (approx. 100 ng/μl ) 7.5 μl PfuTurbo (2.5u/μl), (Stratagene, Catalog #600250)

[0577] 15 μl mixed primer pair (100 ng/μl each) (oligonucleotides a andb, described above)

[0578] 4 μl 100 mM dNTP

[0579] 416 μl H₂O

[0580]500 μl total

[0581] and carrying out the amplification under the following conditionsusing a Stratagene Robocycler 96 hot top thermal cycler: Window 1 95° C.1 minute 1 cycle Window 2 95° C. 1 minute 50° C. 1 minute 30 cycles 72°C. 3 minutes

[0582] The PCR products from each of the five reactions were combinedinto one tube, purified using StrataPrep PCR and eluted in 50 μl 1 mMTris-HCl pH 8.6. The FEN-1 PCR product was analyzed on a gel and wasdetermined to be approximately 1000 bp.

[0583] The PCR product comprising the fen-1 gene was cloned into thepCALnEK LIC vector (Stratagene) by creating ligation independent cloningtermini (LIC), annealing the PCR product comprising the fen-1 gene tothe pCALnEK LIC vector (Stratagene), and transforming cells with theannealing mixture according to the following method. Briefly, followingPCR amplification, the PCR product is purified and treated with Pfu DNApolymerase in the presence of dATP (according to the manual includedwith the Affinity® Protein Expression and Purification System,Stratagene, catalog #200326). In the absence of dTTP, dGTP and dCTP, the3′ to 5′-exonuclease activity of Pfu DNA polymerase removes at least 12and 13 nucleotides at the respective 3′ ends of the PCR product. Thisactivity continues until the first adenine is encountered, producing aDNA fragment with 5′-extended single-stranded tails that arecomplementary to the single-stranded tails of the pCAL-n-EK vector.

[0584] Creating LIC termini

[0585] LIC termini were created by preparing the following mixture:

[0586] 45 μl purified PCR product (˜0.5 μg/μl)

[0587] 2.5 μl 10 mM dATP

[0588] 5 μl 10×cPfu buffer

[0589] 1 μl cPfu (2.5 u/μl)

[0590] 0.5 μl H₂O

[0591] cPfu and cPfu buffer can be obtained from Stratagene (cPfu,Stratagene Catalog #600153 and cPfu buffer, Stratagene Catalog #200532).

[0592] Samples were incubated at 72° C. for 20 minutes and products werecooled to room temperature. To each sample was added 40 ng preparedpCALnEK LIC vector (the prepared vector is available commercially fromStratagene in the Affinity LIC Cloning and Protein Purification Kit(214405)). The vector and insert DNA are combined, allowed to anneal atroom temperature and transformed into highly competent bacterial hostcells (Wyborski et al., 1997, Strategies, 10:1).

[0593] Preparing Cells for Production of FEN

[0594] Two liters of LB-AMP was inoculated with 20 ml of an overnightculture of a FEN-1 clone (clone 3). Growth was allowed to proceed forapproximately 11 hours at which point cells had reached an OD₆₀₀=0.974.Cells were induced overnight (about 12 hours) with 1 mM IPTG. Cells werecollected by centrifugation and the resulting cell paste was stored at−20° C.

[0595] Purification of Tagged FEN-1

[0596] Cells were resuspended in 20 ml of Calcium binding buffer

[0597] CaCl₂ binding Buffer

[0598] 50 mM Tris-HCl (pH 8.0)

[0599] 150 mM NaCl

[0600] 1.0 mM MgOAc

[0601] 2 mM CaCl₂

[0602] The samples were sonicated with a Branson Sonicator using amicrotip. The output setting was 5 and the duty cycle was 90%. Sampleswere sonicated three times and allowed to rest on ice during theintervals. The sonicate was centrifuged at 26,890×g. Clearedsupernatants were mixed with 1 ml of washed (in CaCl₂ binding buffer)calmodulin agarose (CAM agarose) in a 50 ml conical tube and incubatedon a slowly rotating wheel in a cold room (4° C.) for 5 hours. The CAMagarose was collected by light centrifugation (5000 rpm in a table topcentrifuge).

[0603] Following removal of the supernatant, the CAM agarose was washedwith 50 ml CaCl₂ binding buffer and transferred to a disposable dripcolumn. The original container and pipet were rinsed thoroughly toremove residual agarose. The column was rinsed with approximately 200 mlof CaCl₂ binding buffer.

[0604] Elution was carried out with 10 ml of 50 mM NaCl elution buffer(50 mM NaCl, 50 mM Tris-HCl pH 8.0, 2 mM EGTA). 0.5 ml fractions werecollected. A second elution step was carried out with 1M NaCl elutionbuffer wherein 0.5 ml fractions were collected.

[0605] Evaluation of Purified Tagged FEN-1

[0606] Fractions containing CBP-tagged Pfu FEN-1 eluted in 1M NaCl wereboiled in SDS and analyzed by SDS-PAGE on a 4-20% gel stained with SyproOrange (FIG. 5).

[0607] The protein concentration of uncleaved FEN-1 was determined to beapproximately 150 ng/microliter (below).

[0608] Enterokinase Protease (EK) Cleavage of the Purified FEN-1

[0609] Fractions 3-9 were dialyzed in 50 mM NaCl, 50 mM Tris-HCl pH 8.0and 2 mM CaCl₂ overnight at 4° C.

[0610] An opaque, very fine precipitate appeared in the dialyzed FEN-1.When the sample was diluted 1/20 the precipitate was removed. When thesample was diluted 1/3 insoluble material was still detectable. The 1/3diluted material was heated at 37° C. for 2 minutes and mixed with Tween20 to a final concentration of 0.1%. Upon the addition of the Tween 20,there was an almost immediate formation of “strings” and much coarsersolids in the solution which could not be reversed even after thesolution was adjusted to 1M NaCl.

[0611] EK cleavage was carried out using as a substrate the sample thatwas diluted 1/20 as well as with a dilute sample prepared by rinsing thedialysis bag with 1×EK buffer.

[0612] EK cleavage was carried out by the addition of 1 μl EK (1 u/μl)overnight at room temperature (about 16 hours).

[0613] 100 μl of STI agarose combined with 100 μl of CAM agarose wererinsed twice with 10 ml of 1×STI buffer (50 mM Tris-HCl pH 8.0, 200 mMNaCl, 2 mM CaCl₂, 0.1% Tween 20). NaCl was added to the two EK samplesto bring the final concentration to 200 mM NaCl. The two samples werecombined and added to the rinsed agarose. The samples were rotatedslowly on a wheel at 4° C. for three hours and separated by lightcentrifugation in a table top centrifuge (as described). The supernatantwas removed and the resin was rinsed twice with 500 μl 1×STI. The tworinses were combined and saved separately from the original supernatant.Samples were analyzed by SDS-PAGE on a 4-20% gel.

[0614] The concentration of digested product was approximately 23 ng/μlas determined by comparison to a Pfu standard at a concentration ofapproximately 50 ng/ml.

Example 10

[0615] FEN Nuclease Activity

[0616] The endonuclease activity of a FEN nuclease and the cleavagestructure requirements of a FEN nuclease prepared as described inExample 2 can be determined according to the methods described either inthe section entitled “FEN nucleases” or below.

[0617] Briefly, three templates (FIG. 2) are used to evaluate theactivity of a FEN nuclease according to the invention. Template 1 is a5′³³P labeled oligonucleotide (Heltest4) with the following sequence:

5′AAAATAAATAAAAAAAATACTGTTGGGAAGGGCGATCGGTGCG3′.

[0618] The underlined section of Heltest4 represents the regioncomplementary to M13mpl8+. The cleavage product is an 18 nucleotidefragment with the sequence AAAATAAATAAAAAAAAT. Heltest4 binds to M13 toproduce a complementary double stranded domain as well as anon-complementary 5′ overhang. This duplex forms template 2 (FIG. 2).Template 3 (FIG. 2) has an additional primer (FENAS) bound to M13 whichis directly adjacent to Heltest 4. The sequence of FENAS is:5′CCATTCGCCATTCAGGCTGCGCA 3′. In the presence of template 3, a FENnuclease binds the free 5′ terminus of Heltest4, migrates to thejunction and cleaves Beltest4 to produce an 18 nucleotide fragment. Theresulting cleavage products are separated on a 6% acrylamide, 7M ureasequencing gel.

[0619] Templates are prepared as described below: Template 1 Template 2Template 3 Heltest4 14 μl 14 μl 14 μl M13 ** 14 μl 14 μl FENAS ** ** 14μl H₂O 28 μl 14 μl ** 10x Pfu Buff. 4.6 μl 4.6 μl 4.6 μl

[0620] Pfu buffer can be obtained from Stratagene (Catalog #200536).

[0621] The template mixture is heated at 95° C. for five minutes, cooledto room temperature for 45 minutes and stored at 4° C. overnight. Theenzyme samples are as follows:

[0622] A. H₂O (control)

[0623] B. 2 μl undiluted uncleaved FEN-1 (˜445 ng/μl)

[0624] C. 2 μl 1/10 dilution of uncleaved FEN-1 (˜44.5 ng/μl)

[0625] D. 2 μl enterokinase protease (EK) cleaved FEN-1 (˜23 ng/μl)

[0626] The four reaction mixtures are mixed with the three templates asfollows:

[0627] 3 μl template 1, template 2 or template 3

[0628] 0.7 μl 10×cloned Pfu buffer

[0629] 0.6 μl 100 mM MgCl₂

[0630] 2.00 μl FEN-1 or H₂O

[0631] 0.7 μl H₂O

[0632]7.00 μl total volume

[0633] The reactions are allowed to proceed for 30 minutes at 50° C. andstopped by the addition of 2 μl formamide “Sequencing Stop” solution toeach sample. Samples are heated at 95° C. for five minutes and loaded ona 6% acrylamide 7M urea CastAway gel (Stratagene).

[0634] Alternatively, FEN nuclease activity can be analyzed in thefollowing buffer wherein a one hour incubation time is utilized.

[0635] 10×FEN Nuclease Buffer

[0636] 500 mM Tris-HCl pH 8.0

[0637] 100 mM MgCl₂

[0638] The reaction mixture is as follows:

[0639] 3 μl template 1, template 2 or template 3

[0640]0.7 μl 10×FEN nuclease buffer

[0641] 2.00 μl FEN-1 or H₂O (A-D, above)

[0642] 1.3 μl H₂O

[0643] 7.00 μl total volume

[0644] Samples are incubated for one hour at 50° C. in the Robocyler 96hot top thermal cycler. Following the addition of 2 μl of SequencingStop (95% formamide, 20 mM EDTA, 0.05% bromophenol blue, 0.05% xylenecyanol, available from Stratagene) dye solution, samples are heated at99° C. for five minutes. Samples are loaded on an eleven inch long,hand-poured, 20% acrylamide/bis acrylamide, 7M urea gel. The gel is runat 20 watts until the bromophenol blue has migrated approximately ⅔ thetotal distance. The gel is removed from the glass plates and soaked for10 minutes in fix solution (15% methanol, 5% acetic acid) and then for10 minutes in water. The gel is placed on Whatmann 3 mm paper, coveredwith plastic wrap and dried for 2 hours in a heated vacuum gel dryer(˜80° C.). The gel is exposed overnight to X-ray film.

[0645] An autoradiograph of a FEN-1 nuclease assay wherein templates 1,2 and 3 (prepared as described above) are cleaved by the addition of:

[0646] A. H₂O

[0647] B. 2 μl of CBP-tagged Pfu FEN-1

[0648] C. 2 μl of CBP-tagged Pfu FEN-1 diluted (1:10)

[0649] D. 2 μl of EK cleaved Pfu FEN-1

[0650] is presented in FIG. 6.

[0651] The lanes are as follows. Lanes 1A, 1B, 1C and 1D representtemplate 1 cleaved with H₂O, undiluted CBP-tagged Pfu FEN-1, a 1:10dilution of CBP-tagged Pfu FEN-1 and EK cleaved Pfu FEN-1, respectively.Lanes 2A, 2B, 2C and 2D represent template 2 cleaved with H₂O, undilutedCBP-tagged Pfu FEN-1, a 1:10 dilution of CBP-tagged Pfu FEN-1 and EKcleaved Pfu FEN-1, respectively. Lanes 3A, 3B, 3C and 3D representtemplate 3 cleaved with H₂O, undiluted CBP-tagged Pfu FEN-1, a 1:10dilution of CBP-tagged Pfu FEN-1 and EK cleaved Pfu FEN-1, respectively.

[0652] Tagged Pfu FEN-1 contains the N-terminal CBP affinitypurification tag. Any differences in activity between tagged anduntagged versions of FEN-1 are due to differences in proteinconcentration (concentrations of enzyme samples are provided above)since the amounts of tagged versus untagged FEN-1 are not equivalent.Both tagged and untagged Pfu FEN-1 demonstrate cleavage activity.

[0653]FIG. 6 demonstrates the background level of cleavage in theabsence of FEN-1 (lanes 1A, 2A and 3A). Further, this figuredemonstrates that tagged Pfu FEN-1 cleaves more of template 2 ascompared to template 1. In particular, the greatest amount of template 2is cleaved in the presence of undiluted, tagged Pfu FEN-1 (lane 2B).Analysis of template 3 demonstrates that the greatest amount of template3 is cleaved by undiluted, tagged Pfu FEN-1 and the least amount oftemplate 3 is cleaved by diluted tagged FEN-1. Labeled probe migrates asa 40-43 nucleotide band. FEN-1 preferentially cleaves template 3 (whichcomprises an upstream primer) as compared to template 2. The cleavageproduct bands are the major bands migrating at 16-20 nucleotides.Heterogeneity in the labeled cleavage products is the result ofheterogeneity in the labeled substrate, which was not gel-purified priorto use.

Example 11

[0654] PCR Amplification and Detection of β-actin in the Presence of aFEN-1 Nuclease and a Tag Polymerase Deficient in 5′ to 3′ ExonucleaseActivity

[0655] A PCR assay is used to detect a target nucleic acid. According tothe method of this assay, a PCR reaction is carried out in the presenceof a probe having a secondary structure that changes upon binding to atarget nucleic acid and comprising a binding moiety or a tag, Taqpolymerase deficient in 5′ to 3′ exonuclease activity (for example Yaqexo-), and a thermostable FEN-1 nuclease (e.g. Pfu FEN-1, prepared asdescribed in Example 2). Detection of the release of fluorescentlylabeled fragments that bind, via binding of the binding moiety or tag,to a capture element on a solid support indicates the presence of thetarget nucleic acid.

[0656] Duplicate PCR reactions containing 1×Sentinel Molecular beaconcore buffer, 3.5 mM MgCl₂, 200 μM of each dNTP, a Taq polymerasedeficient in 5′ to 3′ exonuclease activity (˜1.45 U), Pfu FEN-1 (˜23ng), β-Actin primers (300 nM each) and a β-actin specific fluorogenicprobe having a secondary structure that changes upon binding of theprobe to the β-Actin target sequence and comprising a binding moiety ortag. 10 ng of human genomic DNA (Promega) is used as the target nucleicacid in each reaction. This reaction is performed in a 50 μl volume.Negative control reactions containing either Pfu FEN-1 alone, a Taqpolymerase deficient in 5′ to 3′ exonuclease activity alone or reactionmixtures containing all components except a human genomic DNA templateare prepared. Positive control reactions comprising 2.5 Units of Taq2000 are also prepared. During the PCR reaction, there is simultaneousformation of a cleavage structure, amplification of the β-actin targetsequence and cleavage of the cleavage structure. Thermocyclingparameters are selected such that cleavage of the cleavage structure isperformed at a cleaving temperature, and the secondary structure of theprobe, when not bound to the target nucleic acid is stable at or belowthe cleaving temperature. Reactions are assayed in a spectrofluorometricthermocycler (ABI 7700). Thermocycling parameters are 95° C. for 2 minand 40 cycles of 95° C. for 15 sec, 60° C. for 60 sec and 72° C. for 15sec. Samples are interrogated during the annealing step.

[0657] Released, fluorescently labeled fragments are bound, via thebinding moiety or tag present on the probe, to a capture element boundto a solid support.

Example 12

[0658] PCR Amplification and Detection of β-actin in the Presence of aFEN-1 Nuclease and a Pfu Polymerase Deficient in 5′ to 3′ ExonucleaseActivity

[0659] A PCR assay is used to detect a target nucleic acid. According tothe method of this assay, a PCR reaction is carried out in the presenceof a probe having a secondary structure that changes upon binding of theprobe to the β-actin target nucleic acid and comprising a binding moietyor tag, Pfu polymerase (naturally lacking 5′ to 3′ exonuclease activity)or, in addition, Pfu polymerase deficient in 3′ to 5′ exonucleaseactivity as well (for example exo- Pfu), and a thermostable FEN-1nuclease (Pfu FEN-1). Detection of the release of fluorescently labeledfragments that bind, via binding of the binding moiety or tag, to acapture element on a solid support indicates the presence of the targetnucleic acid.

[0660] Duplicate PCR reactions containing 1×Cloned Pfu buffer (availablefrom Stratagene, Catalog #200532), 3.0 mM MgCl₂, 200 μM of each dNTP, 5units of a Pfu polymerase deficient in 3′ to 5′ exonuclease activity,tagged or untagged Pfu FEN-1 (˜23 ng), PEF (1 ng) (described in WO98/42860), β-Actin primers (300 nM each), and fluorogenic probe having asecondary structure that changes upon binding of the probe to the targetβ-actin nucleic acid sequence are prepared. 10 ng of human genomic DNA(Promega) is used as the target nucleic acid in each reaction. Reactionsare performed in a 50 μl volume. Negative control reactions comprising aPfu polymerase deficient in both 5′ to 3′ and 3′ to 5′ exonucleaseactivities alone or containing all of the components except the humangenomic DNA template are also prepared. A reaction mixture containing2.5 Units of Taq 2000 is prepared and used as a positive control. Duringthe PCR reaction, there is simultaneous formation of a cleavagestructure, amplification of the β-actin target sequence and cleavage ofthe cleavage structure. Thermocycling parameters are selected such thatcleavage of the cleavage structure is performed at a cleavingtemperature, and the secondary structure of the probe, when not bound tothe target nucleic acid is stable at or below the cleaving temperature.Reactions are analyzed in a spectrofluorometric thermocycler (ABI 7700).Thermocycling parameters are 95° C. for 2 min and 40 cycles of 95° C.for 15 sec, 60° C. for 60 sec and 72° C. for 15 sec.

[0661] Released, fluorescently labeled fragments are bound via thebinding moiety or tag present on the probe, to a capture element boundto a solid support.

Example 13

[0662] An assay according to the invention involving rolling circleamplification is performed using the human ornithine transcarbamylasegene as a target, which is detected in human DNA extracted from buffycoat by standard procedures. Target (400 ng) is heat-denatured for 4minutes at 97° C., and incubated under ligation conditions in thepresence of two 5′-phosphorylated oligomncleotides, an open circle probeand one gap oligonucleotide. The open circle probe has the sequence:gaggagaataaaagtttctcataagactcgtcatgtctcagcagcttctaacggtcactaatacgactcactataggttctgcctctgggaacac,the gap nucleotide for the wild-type sequence is: tagtgatc. FIGS. 7 and8 depict rolling circle probes and rolling circle amplification. Thereaction buffer (40 ul) contains 5 units/μl of T4 DNA ligase (NewEngland Biolabs), 10 mM Tris-HCl, pH 7.5, 0.2 M NaCl, 10 mM MgCl₂, 4 mMATP, 80 nM open circle probe and 100 nM gap oligonucleotide. Afterincubation for 25 minutes at 37° C., 25 ul are removed and added to 25ul of a solution containing 50 mM Tris-HCl, pH 7.5, 10 mM MgCl₂, 1 mMDTT, 400 μM each of dTTP, dATP, dGTP, dCTP, 0.2 μM rolling circlereplication primer: gctgagacatgacgagtc, phi29 DNA polymerase (160 ng/50ul). The sample is incubated for 30 minutes at 30° C.

[0663] RNA is produced from a T7 promoter present in the open circleprobe, by the addition of a compensating buffer (a stock solution orconcentrate) that is diluted to achieve the following concentration ofreagents: 35 mM Tris-HCl, pH 8.2, 2 mM spermidine, 18 mm MgCl₂, 5 mMGMP, 1 mM of ATP, CTP, GTP, 333 uM UTP, 667 uM Biotin-16-UTP, 0.03%Tween 20, 2 units per ul of T7 RNA polymerase. RNA production isperformed as described in U.S. Pat. No. 5,858,033. The incubation isallowed to proceed for 90 minutes at 37° C.

[0664] Five μl of each sample (the actual test sample, a (−) ligasecontrol sample, a (−) phi29 DNA polymerase control and a (−) T7 RNApolymerase control) in duplicate are removed for detection. The reversetranscription process includes the steps of A) ligating the open circle,B) synthesizing rolling circle single stranded DNA, C) making RNA (froma T7 promoter present in the open circle probe), D) reverse transcribingthe RNA to make cDNA, and E) performing PCR amplification of the cDNAusing primers and probes for generation of an detection of FEN cleavagestructures, according to the invention. For reverse transcription, thereagents and protocols supplied with the Stratagene Sentinel Single-TubeRT-PCR Core Reagent Kit (Cat #600505) are used, except for thesubstitution of equal amounts of Yaq DNA polymerase for the Taq 2000 DNApolymerase which is recommended by the manufacturer. Each reactioncontains 1×Sentinel molecular beacon RT-PCR core buffer, 3.5 mM MgCl₂,200 μM of each dNTP, 5 units exo- Pfu, 23 ng Pfu FEN-1, 1 ng PEF, 500 μMeach of the upstream primer: aagtttctcataagactcgtcat, the reverseprimer: aggcagaacctatagtgagtcgt, and the fluorogenic probe (for examplelabeled with FAM-DABCYL) having a secondary structure, as definedherein, that changes upon binding to the target nucleic acid and furthercomprising a binding moiety. The reactions are subjected to incubationfor 30 minutes at 45° C., 3 minutes at 95° C., followed by one cycle ina thermal cycler: 2 minutes at 95° C., 1 minute at 50° C., 1 minute at72° C. The fluorescence in then determined in a fluorescence platereader, such as Stratagene's FluorTracker or PE Biosystems' 7700Sequence Detection System in Plate-Read Mode.

[0665] A crosscheck for the efficiency of detection is possible becauseof the incorporation of Biotin-16-UTP in the rolling circleamplification RNA product. An aliquot of the reactions is captured onglass slides (or alternatively in microwell plates) using an immobilizedcapture probe. Detection of the captured RNA amplicon is described indetail in U.S. Pat. No. 5,854,033, hereby incorporated by reference.

OTHER EMBODIMENTS

[0666] Other embodiments will be evident to those of skill in the art.It should be understood that the foregoing detailed description isprovided for clarity only and is merely exemplary. The spirit and scopeof the present invention are not limited to the above examples, but areencompassed by the following claims.

1. A method of generating a signal indicative of the presence of atarget nucleic acid in a sample, comprising forming a cleavage structureby incubating a sample comprising a target nucleic acid with a probecomprising a binding moiety and a secondary structure that changes uponbinding of said probe to said target nucleic acid, and cleaving saidcleavage structure with a nuclease at a cleaving temperature to releasea nucleic acid fragment and generate a signal, wherein said secondarystructure of said probe is stable at or below said cleaving temperaturewhen not bound to said target nucleic acid, and generation of saidsignal is indicative of the presence of a target nucleic acid in saidsample; and detecting and/or measuring said signal.
 2. The method ofclaim 1 wherein said binding moiety is a tag.
 3. The method of claim 1wherein said binding moiety is a nucleic acid sequence that binds to acapture element.
 4. A method of detecting or measuring a target nucleicacid comprising the steps of: forming a cleavage structure by incubatinga sample containing a target nucleic acid with a probe comprising abinding moiety and a secondary structure that changes upon binding ofsaid probe to said target nucleic acid, cleaving said cleavage structurewith a nuclease at a cleavage temperature to release a nucleic acidfragment, wherein said secondary structure of said probe is stable at orbelow said cleaving temperature when not bound to said target nucleicacid; and detecting and/or measuring the amount of said fragmentcaptured by binding of said binding moiety to a capture element on asolid support as an indication of the presence of the target sequence inthe sample.
 5. The method of claim 4 wherein said binding moiety is atag.
 6. The method of claim 4 wherein said binding moiety is a nucleicacid sequence that binds to said capture element.
 7. The method of claim1 or 4, further comprising a nucleic acid polymerase.
 8. The method ofclaim 1 or 4 wherein said cleavage structure further comprises a 5′flap.
 9. The method of claim 1 or 4 wherein said cleavage structurefurther comprises an oligonucleotide primer.
 10. The method of claim 1or 4 wherein said secondary structure is selected from the groupconsisting of a stem-loop structure, a hairpin structure, an internalloop, a bulge loop, a branched structure, a pseudoknot structure or acloverleaf structure.
 11. The method of claim 1 or 4 wherein saidnuclease is a FEN nuclease.
 12. The method of claim 1 or 4 wherein saidprobe further comprises at least one labeled moiety capable of providinga signal.
 13. The method of claim 1 or 4 wherein a cleavage structure isformed comprising a pair of interactive signal generating labeledmoieties effectively positioned on said probe to quench the generationof a detectable signal when the probe is not bound to said targetnucleic acid.
 14. The method of claim 13 wherein said labeled moietiesare separated by a site susceptible to nuclease cleavage, therebyallowing the nuclease activity of said nuclease to separate the firstinteractive signal generating labeled moiety from the second interactivesignal generating labeled moiety by cleaving at said site susceptible tonuclease cleavage, thereby generating a detectable signal.
 15. Themethod of claim 13 wherein said pair of interactive signal generatingmoieties comprises a quencher moiety and a fluorescent moiety.
 16. Themethod of claim 1 or 4, wherein the probe further comprises a reporter.17. The method of claim 16, wherein the reporter comprises a tag. 18.The method of claim 2 or 5, wherein said fragment is captured by bindingof said tag to a capture element.
 19. The method of claim 17 whereinsaid fragment is captured by binding of said tag to a capture element.20. A polymerase chain reaction process for detecting a target nucleicacid in a sample comprising: providing a cleavage structure comprising aprobe comprising a binding moiety and a secondary structure that changesupon binding of said probe to said target nucleic acid, and a set ofoligonucleotide primers wherein a first primer contains a sequencecomplementary to a region in one strand of said target nucleic acid andprimes the synthesis of a complementary DNA strand, and a second primercontains a sequence complementary to a region in a second strand of thetarget nucleic acid and primes the synthesis of a complementary DNAstrand; and amplifying the target nucleic acid employing a nucleic acidpolymerase as a template-dependent polymerizing agent under conditionswhich are permissive for PCR cycling steps of (i) annealing of primersrequired for amplification to a template nucleic acid sequence containedwithin said target nucleic acid, (ii) extending the primers such thatsaid nucleic acid polymerase synthesizes a primer extension product, and(iii) cleaving said cleavage structure employing a nuclease as acleavage agent for release of labeled fragments from said cleavagestructure thereby creating detectable labeled fragments; and saidcleaving is performed at a cleaving temperature and said secondarystructure of said second primer target nucleic acid is stable at orbelow said cleaving temperature when not bound to said target nucleicacid; and detecting and/or measuring the amount of released, labeledfragment captured by binding of said binding moiety to a capture elementon a solid support as an indicator of the presence of the targetsequence in the sample.
 21. The polymerase chain reaction process ofclaim 20 wherein said nuclease is a FEN nuclease.
 22. The polymerasechain reaction process of claim 20 wherein said binding moiety is a tag.23. The polymerase chain reaction process of claim 20 wherein saidbinding moiety is a nucleic acid sequence that binds to said captureelement.
 24. The polymerase chain reaction process of claim 20 whereinsaid oligonucleotide primers of step b are oriented such that theforward primer is located upstream of said cleavage structure and thereverse primer is located downstream of said cleavage structure.
 25. Thepolymerase chain reaction process of claim 20 wherein the nucleic acidpolymerase has strand displacement activity.
 26. The polymerase chainreaction process of claim 20 wherein the nucleic acid polymerase isthermostable.
 27. The polymerase chain reaction process of claim 20wherein the nuclease is thermostable.
 28. The polymerase chain reactionprocess of claim 20 wherein the nuclease is a flap-specific nuclease.29. The polymerase chain reaction process of claim 20 wherein thelabeled cleavage structure is formed by the addition of at least onelabeled moiety capable of providing a signal.
 30. The polymerase chainreaction process of claim 20 wherein a cleavage structure is formedcomprising a pair of interactive signal generating labeled moietieseffectively positioned on said probe to quench the generation of adetectable signal when said probe is not bound to said target nucleicacid.
 31. The polymerase chain reaction process of claim 20 wherein saidlabeled moieties are separated by a site susceptible to nucleasecleavage, thereby allowing the nuclease activity of said nuclease toseparate the first interactive signal generating labeled moiety from thesecond interactive signal generating labeled moiety by cleaving at saidsite susceptible to nuclease cleavage, thereby generating a detectablesignal.
 32. The method of claim 30 or 31 wherein said pair ofinteractive signal generating moieties comprises a quencher moiety and afluorescent moiety.
 33. The polymerase chain reaction process of claim20, wherein the probe further comprises a reporter.
 34. The polymerasechain reaction process of claim 33, wherein the reporter comprises atag.
 35. The polymerase chain reaction process of claim 22 or 34,wherein said fragment is captured by binding of said tag to a captureelement.
 36. A polymerase chain reaction process for simultaneouslyforming a cleavage structure, amplifying a target nucleic acid in asample and cleaving said cleavage structure comprising the steps of: (a)providing an upstream oligonucleotide primer complementary to a firstregion in one strand of the target nucleic acid, a downstream labeledprobe complementary to a second region in the same strand of the targetnucleic acid, wherein said downstream labeled probe is capable offorming a secondary structure that changes upon binding of said probe tosaid target nucleic acid and, said probe further comprises a bindingmoiety, and a downstream oligonucleotide primer complementary to aregion in a second strand of the target nucleic acid; and providing thatthe upstream primer primes the synthesis of a complementary DNA strand,and the downstream primer primes the synthesis of a complementary DNAstrand; and (b) detecting a nucleic acid which is produced and capturedby binding of said binding moiety to a capture element on a solidsupport in a reaction comprising amplification of said target nucleicacid and cleavage thereof wherein a nucleic acid polymerase is atemplate-dependent polymerizing agent under conditions which arepermissive for PCR cycling steps of (i) annealing of primers to a targetnucleic acid, (ii) extending the primers of step (a), such that saidnucleic acid polymerase synthesizes primer extension products, and saidprimer extension product of the upstream primer of step (a) partiallydisplaces the downstream probe of step (a) to form a cleavage structure;and (iii) cleaving said cleavage structure employing a nuclease as acleavage agent for release of labeled fragments from said cleavagestructure, and said cleaving is performed at a cleaving temperature andsaid secondary structure of said probe target nucleic acid is stable ator below said cleaving temperature when not bound to said target nucleicacid, thereby creating detectable labeled fragments.
 37. The polymerasechain reaction process of claim 36 wherein said cleavage structurefurther comprises a 5′ flap.
 38. A method of forming a cleavagestructure comprising the steps of: (a) providing a target nucleic acid,(b) providing an upstream nucleic acid complementary to said targetnucleic acid, (c) providing a downstream probe having a secondarystructure that changes upon binding of said probe to said target nucleicacid and said probe further comprises a binding moiety; and (d)annealing said target nucleic acid, said upstream nucleic acid and saiddownstream probe; and wherein said cleavage structure can be cleavedwith a nuclease at a cleaving temperature, and said secondary structureof said probe is stable at or below said cleaving temperature when notbound to said target nucleic acid.
 39. The method of claim 38 whereinthe cleavage structure comprises a 5′ flap.
 40. A composition comprisinga target nucleic acid, a probe comprising a binding moiety and asecondary structure that changes upon binding of said probe to a targetnucleic acid, and a nuclease; and wherein said probe and said targetnucleic acid together form a cleavage structure that is cleaved by saidnuclease at a cleaving temperature, and providing that said secondarystructure of said probe is stable at or below said cleaving temperaturewhen not bound to said target nucleic acid.
 41. The composition of claim40 further comprising an oligonucleotide primer.
 42. A kit forgenerating a signal indicative of the presence of a target nucleic acidin a sample, comprising a probe comprising a binding moiety and asecondary structure that changes upon binding of said probe to a targetnucleic acid, and a nuclease; wherein said probe and said target nucleicacid form a cleavage structure that is cleaved by said nuclease at acleaving temperature, and providing that said secondary structure ofsaid probe is stable at or below said cleaving temperature when notbound to said target nucleic acid.
 43. The kit of claim 42 furthercomprising an oligonucleotide primer.
 44. The kit of claim 42, whereinsaid nuclease is a FEN nuclease.
 45. The kit of claim 42, wherein saidprobe comprises at least one labeled moiety.
 46. The kit of claim 42,wherein said probe comprises a pair of interactive signal generatinglabeled moieties effectively positioned to quench the generation of adetectable signal when said probe is not bound to said target nucleicacid.
 47. The kit of claim 46 wherein said labeled moieties areseparated by a site susceptible to nuclease cleavage, thereby allowingthe nuclease activity of said nuclease to separate the first interactivesignal generating labeled moiety from the second interactive signalgenerating labeled moiety by cleaving at said site susceptible tonuclease cleavage, thereby generating a detectable signal.
 48. The kitof claim 46 or 47, wherein said pair of interactive signal generatingmoieties comprises a quencher moiety and a fluorescent moiety.